Estrogen therapy for brain gray matter atrophy and associated disability

ABSTRACT

Provided are methods for slowing, halting, and reversing gray matter atrophy and progression of disability in certain neurodegenerative diseases, including multiple sclerosis, using estrogen, alone or in combination with another agent.

PRIORITY CLAIM

This application is a § 371 national-stage application based on PCTApplication PCT/US15/047906, filed Sep. 1, 2015, which claims priorityto U.S. Provisional Patent Application No. 62/044,757, filed on Sep. 2,2014, which is hereby incorporated by reference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant NumberNS051591, awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND

Multiple sclerosis (MS) is a chronic, often debilitating diseaseaffecting the central nervous system (CNS, brain and spinal cord). MSaffects more than 1 million people worldwide and is the most commonneurological disease among young adults, particularly women. The exactcause of MS is still unknown. MS is an autoimmune disease in whichmyelin sheaths surrounding neuronal axons are destroyed. This conditioncan cause weakness, impaired vision, loss of balance, poor musclecoordination, and cognitive difficulties.

While MS causes multifocal white matter lesions that are readily visibleon magnetic resonance imaging (MRI), multifocal gray matter lesions,though less conspicuous, are present as well. Gray matter involvementbegins early in MS and is mostly characterized by volume loss, i.e.,cortical thinning and subcortical atrophy. Brain atrophy, and inparticular, brain gray matter atrophy, is now associated with disabilityin MS. Shiee N et al. PLoS ONE 7(5): e37049 (2012).

MS takes several forms, with new symptoms either occurring in isolatedattacks (relapsing forms) or building up over time (progressive forms).Between attacks, symptoms may disappear completely; however, permanentneurological problems often occur, especially as the disease advances.

In 1996, the United States National Multiple Sclerosis Society describedfour clinical subtypes of MS: (i) relapsing-remitting; (ii)secondary-progressive; (iii) primary-progressive; and (iv)progressive-relapsing.

Relapsing-remitting MS is characterized by unpredictable relapsesfollowed by periods of months to years of relative quiet (remission)with no new signs of disease activity. Deficits that occur duringattacks may either resolve or leave sequelae, the latter in about 40% ofattacks and being more common the longer a person has had the disease.This describes the initial course of 80% of individuals with MS. Whendeficits always resolve between attacks, this is sometimes referred toas benign MS, although people will still build up some degree ofdisability in the long term. On the other hand, the term malignantmultiple sclerosis is used to describe people with MS having reachedsignificant level of disability in a short period of time. Therelapsing-remitting subtype usually begins with a clinically isolatedsyndrome (CIS). In CIS, a person has an attack suggestive ofdemyelination but does not fulfill the criteria for multiple sclerosis;30 to 70% of persons experiencing CIS go on to develop MS.

Secondary-progressive MS occurs in around 65% of those with initialrelapsing-remitting MS, who eventually have progressive neurologicdecline between acute attacks without any definite periods of remission.Occasional relapses and minor remissions may appear. The median lengthof time between disease onset and conversion from relapsing-remitting tosecondary progressive MS is 19 years.

Primary-progressive MS occurs in approximately 10-20% of individuals,with no remission after the initial symptoms. It is characterized byprogression of disability from onset, with no, or only occasional andminor, remissions and improvements. The usual age of onset for theprimary progressive subtype is later than of the relapsing-remittingsubtype, but similar to the age that secondary-progressive MS usuallytransitions from relapsing-remitting MS, around 40 years of age.

Progressive-relapsing MS describes those individuals who, from onset,have a steady neurologic decline but also have clear superimposedattacks. This is the least common of all subtypes.

Currently the following agents are approved by the U.S. Food and DrugAdministration (FDA) to reduce disease activity and disease progressionfor many people with relapsing forms of MS, includingrelapsing-remitting MS, as well as secondary-progressive andprogressive-relapsing MS in those people who continue to have relapses:dimethyl fumarate (Tecfidera®; BG-12), fingolimod (Gilenya®), glatirameracetate (Copaxone®), interferon beta-1a (Avonex® and Rebif®), interferonbeta-1b (Betaseron® and Extavia®), mitoxantrone (Novantrone®),natalizumab (Tysabri®), and teriflunomide (Aubagio®). Among drugs underdevelopment for the treatment of MS is anti-LINGO-1 (BIIB033,Biogen-Idec), a fully human monoclonal antibody which targets LINGO-1, aprotein expressed selectively in the CNS that is known to negativelyregulate axonal myelination and axonal regeneration. However, many ofthese therapies fail to successfully treat all patients or all symptomsin treated patients, and many of these therapies are associated withundesirable side effects. Accordingly, alternative therapies are needed.

SUMMARY

An aspect of the invention is a method of slowing or halting gray matteratrophy in a neurodegenerative disease patient, comprising administeringto the patient an estrogen.

An aspect of the invention is a method of treating neurodegenerativedisease in a patient having substantial brain gray matter atrophy,comprising administering to the patient an effective amount of anestrogen. In some aspects, the present invention relates to a method oftreating or preventing a neurodegenerative disease, in a patient havinggreater than about 0.1% brain gray matter loss per annum, comprisingadministering to the patient an effective amount of an estrogen.

An aspect of the invention is a method of treating a neurodegenerativedisease in a patient having a loss of brain gray matter of greater thanabout 0.3% in a period of at least about 6 months, comprisingadministering to the patient an effective amount of an estrogen.

An aspect of the invention is a method of treating a neurodegenerativedisease in a patient having memory loss over a period of at least about6 months, comprising administering to the patient an effective amount ofan estrogen.

An aspect of the invention is a method of treating a neurodegenerativedisease in a patient who is non-responsive to treatment with a multiplesclerosis therapy, comprising administering to the patient an effectiveamount of an estrogen, wherein the patient is classified asnon-responsive based on a substantial loss of brain gray matter volumeduring treatment with the first treatment agent.

An aspect of the invention is a method of treating a neurodegenerativedisease in a patient who is non-responsive to treatment with a multiplesclerosis therapy, comprising administering to the patient an effectiveamount of an estrogen, wherein the patient is classified asnon-responsive based on a substantial loss of memory during treatmentwith the first treatment agent.

An aspect of the invention is a method of reversing gray matter atrophyin a neurodegenerative disease patient, comprising administering to thepatient an estrogen.

An aspect of the invention is a method of slowing or halting progressionof disability in a neurodegenerative disease patient, comprisingadministering to the patient an estrogen.

An aspect of the invention is a method of reversing progression ofdisability in a neurodegenerative disease patient, comprisingadministering to the patient an estrogen.

An aspect of the invention is a method of preventing progression of aneurodegenerative disease, comprising administering to a patientpresenting with brain gray matter atrophy an effective amount of anestrogen.

An aspect of the invention is a method of slowing or halting progressionof memory loss in a neurodegenerative disease patient, comprisingadministering to the patient an estrogen.

An aspect of the invention is a method of reversing progression ofmemory loss in a neurodegenerative disease patient, comprisingadministering to the patient an estrogen.

An aspect of the invention is a method of preventing progression of aneurodegenerative disease, comprising administering to a patientpresenting with memory loss an effective amount of an estrogen.

An aspect of the invention is method of treating and/or slowingprogression of a neurodegenerative disease, comprising: evaluating apatient's brain gray matter; and administering an effective amount of anestrogen to a patient suffering from brain gray matter atrophy.

An aspect of the invention is method of treating and/or slowingprogression of a neurodegenerative disease, comprising: evaluating apatient's memory and administering an effective amount of an estrogen toa patient suffering from memory loss.

In certain embodiments, the estrogen is selected from estriol (E3),estradiol (E2), estrone (E1), an ester thereof, a pharmaceuticallyacceptable salt of an ester thereof, and any combination thereof.

In certain embodiments, the estrogen is estriol.

In certain embodiments, the method further comprises administering tothe patient an immunotherapeutic agent, wherein the immunotherapeuticagent is neither an estrogen nor a progestogen, e.g., animmunotherapeutic agent selected from interferon-beta 1a,interferon-beta 1b, glatiramer acetate, natalizumab, mitoxantrone,fingolimod, teriflunomide, and dimethyl fumarate.

Another aspect of the invention is a method of treating aneurodegenerative disease, comprising

administering to a subject in need thereof a treatment regimen;

assessing a change in total (whole) brain gray matter volume of thesubject over a period of time; and

changing the treatment regimen if the total (whole) brain gray mattervolume decreases by at least about 0.3 percent between a firstassessment and a second assessment.

In certain embodiments, the second assessment occurs at least about 6months after the first assessment, such as about six months after thefirst assessment. In other embodiments, the second assessment occursabout one year after the first assessment, and the treatment regimen ischanged if the total (whole) brain gray matter volume decreases by atleast about 0.6 percent between the first assessment and the secondassessment. In yet other embodiments, the second assessment occurs abouttwo years after the first assessment, and the treatment regimen ischanged if the total (whole) brain gray matter volume decreases by atleast about 1.0 percent between the first assessment and the secondassessment.

In certain embodiments, the assessing comprises performing brainmagnetic resonance imaging (MRI).

In certain embodiments, the assessing consists of performing brainmagnetic resonance imaging (MRI).

In certain embodiments, gray matter is assessed using an imagingtechnique or a surrogate marker.

In certain embodiments, the second assessment is performed at leastabout one year after the first assessment.

In certain embodiments, the second assessment is performed about oneyear after the first assessment.

In certain embodiments, the treatment regimen comprises estrogen,wherein the estrogen is selected from estriol (E3), estradiol (E2),estrone (E1), an ester thereof, a pharmaceutically acceptable salt of anester thereof, and any combination thereof.

In certain embodiments, the estrogen is estriol.

In certain embodiments, the treatment regimen further comprisesadministering to the subject an immunotherapeutic agent.

In certain embodiments, the immunotherapeutic agent is selected frominterferon-beta 1a, interferon-beta 1b, glatiramer acetate, natalizumab,mitoxantrone, fingolimod, teriflunomide, dimethyl fumarate,mycophenolate mofetil, paclitaxel, cyclosporine, corticosteroids,azathioprine, cyclophosphamide, methotrexate, cladribine,4-aminopyridine, and tizanidine.

An aspect of the invention is a method of treating a neurodegenerativedisease, comprising

administering to a human subject in need thereof a treatment regimen;

assessing a change in gray matter volume of at least two brain regionsof the subject over a period of time; and

changing the treatment regimen if the gray matter volume of the at leasttwo brain regions decreases by at least about 0.3 percent between afirst assessment and a second assessment. In certain embodiments, thesecond assessment occurs at least about 6 months after the firstassessment, such as about six months after the first assessment. Inother embodiments, the second assessment occurs about one year after thefirst assessment, and the treatment regimen is changed if the graymatter volume of the at least two brain regions decreases by at leastabout 0.6 percent between the first assessment and the secondassessment. In yet other embodiments, the second assessment occurs abouttwo years after the first assessment, and the treatment regimen ischanged if the gray matter volume of the at least two brain regionsdecreases by at least about 1.0 percent between the first assessment andthe second assessment.

In certain embodiments, the assessing comprises performing brainmagnetic resonance imaging (MRI).

In certain embodiments, the assessing consists of performing brainmagnetic resonance imaging (MRI).

An aspect of the invention is a method of treating a neurodegenerativedisease, comprising: administering to a human subject in need thereof atreatment regimen; assessing a change in memory over a period of time;and changing the treatment regimen if the memory of the subject declinesbetween a first assessment and a second assessment. In certainembodiments, the second assessment occurs at least about 6 months afterthe first assessment, such as about six months after the firstassessment. In other embodiments, the second assessment occurs about oneyear after the first assessment, and the treatment regimen is changed ifthe memory of the subject declines between the first assessment and thesecond assessment. In yet other embodiments, the second assessmentoccurs about two years after the first assessment, and the treatmentregimen is changed if the memory of the subject declines between thefirst assessment and the second assessment.

In certain embodiments, the second assessment is performed at leastabout one year after the first assessment.

In certain embodiments, the second assessment is performed about oneyear after the first assessment.

In certain embodiments, the treatment regimen comprises estrogen,wherein the estrogen is selected from estriol (E3), estradiol (E2),estrone (E1), an ester thereof, a pharmaceutically acceptable salt of anester thereof, and any combination thereof.

In certain embodiments, the estrogen is estriol.

In certain embodiments, the treatment regimen further comprisesadministering to the subject an immunotherapeutic agent.

In certain embodiments, the immunotherapeutic agent is selected frominterferon-beta 1a, interferon-beta 1b, glatiramer acetate, natalizumab,mitoxantrone, fingolimod, teriflunomide, dimethyl fumarate,mycophenolate mofetil, paclitaxel, cyclosporine, corticosteroids,azathioprine, cyclophosphamide, methotrexate, cladribine,4-aminopyridine, and tizanidine.

In certain embodiments, the neurodegenerative disease is multiplesclerosis.

In certain embodiments, the neurodegenerative disease isrelapsing-remitting multiple sclerosis.

In certain embodiments, the neurodegenerative disease issecondary-progressive multiple sclerosis.

In certain embodiments, the neurodegenerative disease isprimary-progressive multiple sclerosis.

In certain embodiments, the neurodegenerative disease isprogressive-relapsing multiple sclerosis.

In certain embodiments, the neurodegenerative disease is clinicallyisolated syndrome (CIS).

In certain embodiments, the subject is a subject being treated with animmunotherapeutic agent yet experiencing a relapse and/or progression ofthe multiple sclerosis.

An aspect of the invention is a method of treating multiple sclerosis ina patient having a loss of brain gray matter of greater than about 0.3%in a period of at least about 6 months, comprising administering to thepatient about 8 mg of estriol daily.

An aspect of the invention is a method of treating multiple sclerosis ina patient having memory loss over a period of at least about 6 months,comprising administering to the patient about 8 mg of estriol daily.

Although the methods disclosed throughout the specification and claimsare useful for treating multiple sclerosis in its various forms andstages, these methods can also be applied the treatment of otherneurodegenerative diseases, such as, by way of illustration, Alzheimer'sdisease, Parkinson's disease, stroke, amyotrophic lateral sclerosis,cerebellar ataxia, frontotemporal dementia, prion disease, Huntington'sDisease, cerebral ischemia, cerebral dementia syndrome,infection-induced neurodegeneration disorders (e.g.,AIDS-encephalopathy, Creutzfeld-Jakob disease, encephalopathies inducedby rubiola and herpes viruses and borrelioses), metabolic-toxicneurodegenerative disorders (such as hepatic-, alcoholic-, hypoxic-,hypo- or hyperglycemically-induced encephalopathies), encephalopathiesinduced by solvents or pharmaceuticals, trauma-induced brain damage, andspinal cord injury. In certain preferred embodiments, theneurodegenerative disease is multiple sclerosis. In preferredembodiments, the patient is a woman. In some embodiments, the patient isa premenopausal or perimenopausal woman. In other embodiments, thepatient is a postmenopausal woman.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 includes two panels, identified as panels (A) and (B). Panel Ashows the disposition of subjects enrolled in a clinical trial ofestriol for treating multiple sclerosis. Panel B shows the study design.“Taper” indicates a period of reduction of either estriol or placeboover the course of 4 weeks at end of study, after month 24 clinic visit.Specifically, the dose of estriol was reduced by half (from 8 mg to 4mg) for 2 weeks, then reduced by half again (from 4 mg to 2 mg) for 2weeks, then discontinued. “x” indicates the administration of aprogestin (0.7 mg norethindrone) orally each day for 2 weeks every threemonths, beginning at study month 6. “o” indicates the administration ofa placebo for the progestin orally each day for 2 weeks every threemonths, beginning at study month 6.

FIG. 2 includes four panels, identified as panels (A), (B), (C), and(D). Each panel corresponds to a bar graph depicting gray matter volumechange in the cerebral cortex (A), thalamus (B), caudate (C), andputamen (D) in five patients treated with estriol plus Copaxone® (blackbars) and five patients treated with placebo plus Copaxone® (gray bars).M12, comparison between month 0 (pre-treatment baseline) and month 12.M24, comparison between month 0 and month 24. Error bars, 1 SD.

FIG. 3 is a bar graph depicting annualized relapse rates with Copaxone®plus estriol treatment as compared to Copaxone® plus estriol placebotreatment.

FIG. 4 is a bar graph depicting Paced Serial Addition Test (PASAT)cognitive test scores for all subjects (All), subjects with baselinescores of less than 55/60 (<55), and subjects with baseline scoresgreater than or equal to 55/60 (≥55). Comparison is made betweentreatment groups receiving Copaxone® plus estriol or Copaxone® plusestriol placebo. Data are expressed as percent change from baseline ineach treatment group.

FIG. 5 is a graph depicting the proportion of all subjects who hadsustained improvement of 3 points in PASAT scores for 6 months.Comparison is made between treatment groups receiving Copaxone® plusestriol (upper curve) or Copaxone® plus estriol placebo (lower curve).

FIG. 6 is a graph depicting Expanded Disability Status Scale (EDSS)scores over 24 months for treatment groups receiving Copaxone® plusestriol (slope=−0.11, p=0.06) or Copaxone® plus estriol placebo(slope=−0.01, p=0.90).

FIG. 7 includes nine panels, identified as panels (A), (B), (C), (D),(E), and (F). Panel (A) shows that EDSS improvement was observed at 24months in the Estriol+GA within group comparison (median=−0.5, P=0.03),with no change in the Placebo+GA group (median=0, P=NS), and betweengroups comparison not reaching significance. (B) PASAT score improvementwas observed at 12 months in the Estriol+GA within group comparison(P=0.005), with no change in the Placebo+GA group, and between groupcomparison significant (P=0.04), however scores assessed at the 24 monthtime point were no different between groups. All data are expressed aschange in mean absolute scores over time as compared to baseline. (C-F)Change in volume from baseline for cortical gray matter in C; for wholegray matter in D; for whole white matter in E; and for whole brain in F.Right: Significant voxel-wise gray matter loss from baseline to month 12was more in Placebo+GA (top left subpanel) than in Estriol+GA (top rightpanel), with regions showing significant between group differencesdemonstrated by intensity heat map (bottom panel). Disabilities areexpressed as means+/−SE. Negative values indicate improvement for EDSSscores. Positive values indicate improvement for PASAT scores.EDSS=Expanded Disability Status Scale; PASAT=Paced Auditory SerialAddition Test (at 3 seconds). Volumes are expressed as mean percentchange+/−SE from baseline. *=P<0.10, **=P<0.05. VBM results arevisualized on the mean template and thresholded at P≤0.05, FDRcorrected. Black indicates Placebo+GA, while Gray indicates Estriol+GA.

FIG. 8 shows MSQOL composite scores for Physical outcomes were improvedin the Estriol+GA group (p=0.02), with no change in the Placebo+GAgroup, between group comparisons not reaching significance. All data areexpressed as change in mean absolute scores over time as compared tobaseline. Positive values indicate improvement for MSQOL Physicalscores.

FIG. 9 shows maximum intensity projections of voxel-wise gray matteratrophy superimposed onto 3 orthogonal planes through the brain. At 12months, significant localized gray matter loss was observed in thePlacebo+GA group as compared to baseline (top) and in the Estriol+GAgroup as compared to baseline (middle), each shown in red in the 3planes. Regions of significantly more gray matter loss in the Placebo+GAgroup as compared to the Estriol+GA group on between group comparisonsare shown in yellow in the 3 planes (bottom). Gray matter loss is alsovisualized as projected onto a surface rendering of the mean template(lower right corner of each panel). All results are corrected formultiple comparisons by controlling the FDR at P≤0.05.

DETAILED DESCRIPTION

Approximately 50% of people diagnosed with multiple sclerosis (MS) willdevelop problems with cognition. Currently, there are no FDA-approvedtreatments targeting cognitive function in MS. Multiple sclerosisrelapses are known to be significantly decreased by approximately 80%during late pregnancy. This disease improvement may be due to estriol,an estrogen unique to pregnancy. Estriol blood levels go fromundetectable levels prior to pregnancy, increase during pregnancy andreach highest levels during late pregnancy. Further, estrogen treatmenthas been shown to have favorable effects on cognition in animal modelsof other neurological diseases.

There is an unmet need for a treatment to halt disability in MS. Braingray matter (GM) atrophy is a biomarker for disability in MS. Atreatment that can halt GM atrophy in MS would be promising to haltdisability in MS. This GM biomarker could be used in trials to screendrugs aiming to halt disability in MS. This GM biomarker could also beused to follow patients to predict which will have future worsening ofdisability and hence may need to change their treatment regimen to startestriol treatment (either as a monotherapy in untreated or as an add-ontreatment in those on standard MS treatment) to prevent further declinefrom occurring or to slow further decline. Also, this GM biomarker couldalso be used to identify patients in early stages of disease andoptionally direct the use of treatment methods described herein.Further, this GM biomarker could also be used to identify patients whohave aggressive (e.g. non-responsive to standard treatments) orlate-stage forms of disease and optionally direct the use of treatmentmethods described herein.

The invention is based, in part, on the surprising discovery by theinventor that an estrogen, at least in combination with a standardtreatment for MS, dramatically slows, halts, and even reverses graymatter atrophy in MS. In accordance with the invention, estrogen is alsobelieved to slow, halt, and even reverse progression of disability in MSand other neurodegenerative diseases.

As will be described in greater detail below, the estrogen can be usedin combination with certain immunotherapeutic agents, for example,disease-modifying therapeutics (DMTs) useful in the treatment MS.

Alternatively or in addition, as will be described in greater detailbelow, the estrogen can be used in combination with a progestogen, forexample where the estrogen is administered continuously whereas theprogestogen is administered from time to time.

An aspect of the invention is a method of slowing or halting gray matteratrophy in a neurodegenerative disease patient, comprising administeringto the patient an estrogen.

An aspect of the invention is a method of treating neurodegenerativedisease in a patient having substantial brain gray matter atrophy,comprising administering to the patient an effective amount of anestrogen. In some aspects, the present invention relates to a method oftreating or preventing a neurodegenerative disease, in a patient havinggreater than about 0.1% brain gray matter loss per annum (e.g., about0.2%, or about 0.3%, or about 0.4%, or about 0.5%, or about 0.6%, orabout 0.7%, or about 0.8%, or about 0.9%, or about 1.0%, or about 1.1%,or about 1.2%, or about 1.3%, or about 1.4%, or about 1.5%, or about2.0% brain gray matter loss per annum), comprising administering to thepatient an effective amount of an estrogen.

An aspect of the invention is a method of treating a neurodegenerativedisease, in a patient having a loss of brain gray matter of greater thanabout 0.3% in a period of at least about 6 months (e.g., about 0.4%, orabout 0.5%, or about 0.6%, or about 0.7%, or about 0.8%, or about 0.9%,or about 1.0%, or about 1.1%, or about 1.2%, or about 1.3%, or about1.4%, or about 1.5%, or about 2.0 in a period of about 6 months, orabout 12 months, or about 18 months, or about 24 months), comprisingadministering to the patient an effective amount of an estrogen.

An aspect of the invention is a method of treating a neurodegenerativedisease, in a patient having memory loss over a period of at least about6 months (e.g., a period of about 6 months, or about 12 months, or about18 months, or about 24 months), comprising administering to the patientan effective amount of an estrogen.

The terms “substantial brain gray matter atrophy” and “substantial lossof brain gray matter volume” as used herein, refers to a patient havinggreater than about 0.5% brain gray matter loss per annum (e.g., about0.5%, or about 0.6%, or about 0.7%, or about 0.8%, or about 0.9%, orabout 1.0%, or about 1.1%, or about 1.2%, or about 1.3%, or about 1.4%,or about 1.5%, or about 2.0% brain gray matter loss per annum). Forexample, “substantial brain gray matter atrophy” and “substantial lossof brain gray matter volume” may refer to a patient having a loss ofbrain gray matter of greater than about 0.3% in a period of at leastabout 6 months (e.g., about 0.4%, or about 0.5%, or about 0.6%, or about0.7%, or about 0.8%, or about 0.9%, or about 1.0%, or about 1.1%, orabout 1.2%, or about 1.3%, or about 1.4%, or about 1.5%, or about 2.0 ina period of about 6 months, or about 12 months, or about 18 months, orabout 24 months). The amount of gray matter loss may be determined, forexample, by using an imaging technique or surrogate marker.

An aspect of the invention is a method of treating a neurodegenerativedisease in a patient who is non-responsive to treatment with a multiplesclerosis therapy (e.g., one or more of dimethyl fumarate (Tecfidera®;BG-12), fingolimod (Gilenya®), glatiramer acetate (Copaxone®),interferon beta-1a (e.g., Avonex® and Rebif®), interferon beta-1b (e.g.,Betaseron® and Extavia®), mitoxantrone (Novantrone®), natalizumab(Tysabri®), teriflunomide (Aubagio®) and anti-LINGO-1 antibody (BIIB033,Biogen-Idec), comprising administering to the patient an effectiveamount of an estrogen, wherein the patient is classified asnon-responsive based on a substantial loss of brain gray matter volumeduring treatment with the first treatment agent.

An aspect of the invention is a method of treating multiple sclerosis ina patient having a loss of brain gray matter of greater than about 0.3%in a period of at least about 6 months, comprising administering to thepatient about 8 mg of estriol daily.

An aspect of the invention is a method of treating multiple sclerosis ina patient having memory loss over a period of at least about 6 months,comprising administering to the patient about 8 mg of estriol daily.

An aspect of the invention is a method of reversing gray matter atrophyin a neurodegenerative disease patient, comprising administering to thepatient an estrogen.

An aspect of the invention is a method of slowing or halting progressionof disability in a neurodegenerative disease patient, comprisingadministering to the patient an estrogen.

An aspect of the invention is a method of reversing memory loss in aneurodegenerative disease patient, comprising administering to thepatient an estrogen.

An aspect of the invention is a method of slowing or halting memory lossin a neurodegenerative disease patient, comprising administering to thepatient an estrogen.

An aspect of the invention is a method of reversing progression ofdisability in a neurodegenerative disease patient, comprisingadministering to the patient an estrogen.

An aspect of the invention is a method of preventing progression of aneurodegenerative disease, comprising administering to a patientpresenting with brain gray matter atrophy an effective amount of anestrogen.

An aspect of the invention is a method of preventing progression of aneurodegenerative disease, comprising administering to a patientpresenting with memory loss an effective amount of an estrogen.

An aspect of the invention is method of treating and/or slowingprogression of a neurodegenerative disease, comprising: evaluating apatient's brain gray matter; and administering an effective amount of anestrogen to a patient suffering from brain gray matter atrophy.

An aspect of the invention is method of treating and/or slowingprogression of a neurodegenerative disease, comprising: evaluating apatient's memory; and administering an effective amount of an estrogento a patient suffering from memory loss.

In certain embodiments, the neurodegenerative disease is Alzheimer'sdisease, Parkinson's disease, multiple sclerosis, stroke, amyotrophiclateral sclerosis, cerebellar ataxia, frontotemporal dementia, priondisease, Huntington's disease, cerebral ischemia, cerebral dementiasyndrome, infection-induced neurodegeneration disorders (e.g.,AIDS-encephalopathy, Creutzfeld-Jakob disease, encephalopathies inducedby rubiola and herpes viruses and borrelioses), metabolic-toxicneurodegenerative disorders (such as hepatic-, alcoholic-, hypoxic-,hypo- or hyperglycemically-induced encephalopathies), encephalopathiesinduced by solvents or pharmaceuticals, trauma-induced brain damage, orspinal cord injury.

In certain embodiments, the neurodegenerative disease is Alzheimer'sdisease, Parkinson's disease, multiple sclerosis, stroke, amyotrophiclateral sclerosis, cerebellar ataxia, frontotemporal dementia, priondisease, Huntington's disease, cerebral ischemia, cerebral dementiasyndrome, infection-induced neurodegeneration disorders (e.g.,AIDS-encephalopathy, Creutzfeld-Jakob disease, encephalopathies inducedby rubiola and herpes viruses and borrelioses), metabolic-toxicneurodegenerative disorders (such as hepatic-, alcoholic-, hypoxic-,hypo- or hyperglycemically-induced encephalopathies), encephalopathiesinduced by solvents or pharmaceuticals, or trauma-induced brain damage.

In certain embodiments, the neurodegenerative disease is multiplesclerosis.

In certain embodiments, the neurodegenerative disease is clinicallyisolated syndrome (CIS).

In certain embodiments, the neurodegenerative disease isrelapsing-remitting multiple sclerosis.

In certain embodiments, the neurodegenerative disease issecondary-progressive multiple sclerosis.

In certain embodiments, the neurodegenerative disease isprimary-progressive multiple sclerosis.

In certain embodiments, the neurodegenerative disease isprogressive-relapsing multiple sclerosis.

The term “estrogen” as used herein refers to any biologically activeform of estrogen or precursor thereof. The term “estrogen” thus embracesnaturally occurring, synthetic, and semi-synthetic forms of estrogen,and biologically active, pharmaceutically acceptable salts and estersthereof. In certain embodiments, estrogen is selected from estriol (E3),estradiol (E2), estrone (E1), an ester thereof, a pharmaceuticallyacceptable salt of an ester thereof, and any combination of theforegoing. In certain embodiments, estrogen is estriol (E3) or an esterthereof, or a pharmaceutically acceptable salt of an ester thereof. Forexample, the estrogen can be estriol, estriol succinate, estrioldihexanoate, or estriol sulfate. In other embodiments, estrogen isestradiol (E2) or an ester thereof, or a pharmaceutically acceptablesalt of an ester thereof, while in yet other embodiments, estrogen isestrone (E1) or an ester thereof, or a pharmaceutically acceptable saltof an ester thereof. In certain preferred embodiments, estrogen isestriol (E3). In certain embodiments, estrogen is estradiol (E2). Incertain embodiments, estrogen is estrone (E1).

In certain embodiments, the estrogen is administered in a dose equal orequivalent to about 200 μg to about 20 mg estriol daily. For example, adose of 2 to 4 mg of estriol is generally considered to be equivalent to0.6 to 1.25 mg of conjugated estrogens or estrone. In certainembodiments, the estrogen is administered in a dose equal or equivalentto about 1 mg to about 10 mg estriol daily, preferably equal orequivalent to about 8 mg estriol daily. In most preferred embodiments,the estrogen is estriol administered in a dose of about 8 mg estrioldaily.

In certain embodiments, the estrogen is formulated for oraladministration, e.g., in a dose equal or equivalent to about 200 μg toabout 20 mg estriol daily. For example, a dose of 2 to 4 mg of estriolis generally considered to be equivalent to 0.6 to 1.25 mg of conjugatedestrogens or estrone. In certain embodiments, the estrogen is formulatedfor oral administration in a dose equal or equivalent to about 1 mg toabout 10 mg estriol daily, preferably equal or equivalent to about 8 mgestriol daily. In most preferred embodiments, the estrogen is estriolformulated for oral administration in a dose of about 8 mg estrioldaily.

In certain embodiments, the estrogen is orally administered in a doseequal or equivalent to about 200 μg to about 20 mg estriol daily. Forexample, a dose of 2 to 4 mg of estriol is generally considered to beequivalent to 0.6 to 1.25 mg of conjugated estrogens or estrone. Incertain embodiments, the estrogen is orally administered in a dose equalor equivalent to about 1 mg to about 10 mg estriol daily, preferablyequal or equivalent to about 8 mg estriol daily. In most preferredembodiments, the estrogen is estriol orally administered in a dose ofabout 8 mg estriol daily.

An “effective amount”, as used herein, refers to an amount that issufficient to achieve a desired biological effect. A “therapeuticallyeffective amount”, as used herein refers to an amount that is sufficientto achieve a desired therapeutic effect. For example, a therapeuticallyeffective amount can refer to an amount that is sufficient to improve atleast one sign or symptom of MS. Alternatively, a therapeuticallyeffective amount can refer to an amount sufficient to halt or slow therate of brain gray matter atrophy that develops over a given timeinterval.

A therapeutically effective dose of the estrogen is, in someembodiments, one sufficient to raise the serum concentration above basallevels, and preferably to pregnancy levels or above pregnancy levels. Incertain embodiments, the therapeutically effective dose of the estrogenis selected to result in serum levels in a patient equivalent to thesteroid hormone level of that agent in women in the second or thirdtrimester of pregnancy.

For example, during the normal female menstrual cycle estradiol levelsare in the range of about 350 pg/mL serum. During pregnancy, there isabout a 100-fold increase in the level of estradiol to about 10,000 toabout 35,000 pg/mL serum. Correale et al., J Immunol 161:3365-74 (1998)and Gilmore et al., J Immunol 158:446-51 (1997). In contrast, estriollevels are undetectable during the menstrual cycle in the non-pregnantstate. Estradiol levels rise progressively during pregnancy to levelsfrom 3,000 to 30,000 pg/mL (3 to 30 ng/mL).

In one embodiment, where the estrogen is estriol, the dose is from about4 to 16 milligrams daily, and more specifically, about 8 milligramsdaily. In this embodiment, blood serum levels preferably reach at leastabout 2 ng/mL, may reach about 10 to about 35 ng/mL, or most preferablyabout 20-30 ng/mL. Sicotte et al. Neurology 56:A75 (2001). In someembodiments, estradiol (E2) levels would preferably reach at least about2 ng/mL and most preferably about to 10-35 ng/mL. In some embodiments,estrone (E1) levels would preferably reach at least about 2 ng/mL andmost preferably about 5-18 ng/mL. DeGroot et al., Endocrinology3(9):2171-223 (1994).

The dosage of the estrogen may be selected for an individual patientdepending upon the route of administration, severity of disease, age andweight of the patient, other medications the patient is taking and otherfactors normally considered by the attending physician, when determiningthe individual regimen and dosage level as the most appropriate for aparticular patient. Furthermore, the exact individual dosages can beadjusted somewhat depending on a variety of factors, including thespecific combination of the agents being administered, the time ofadministration, the route of administration, the nature of theformulation, the rate of excretion, the particular disease beingtreated, the severity of the disorder, and the anatomical location ofthe disorder. Some variations in the dosage can be expected. In vitro orin vivo assays can be employed to help identify optimal dosage ranges.

A therapeutically effective dose of the estrogen included in the dosageform is selected at least by considering the type of estrogen selectedand the mode of administration. The dosage form may include the estrogenin combination with other inert ingredients, including adjuvants andpharmaceutically acceptable carriers for the facilitation of dosage tothe patient as known to those skilled in the pharmaceutical arts. Thedosage form may be any form suitable to cause the estrogen to enter intothe tissues of the patient.

Pharmaceutically acceptable carriers can optionally comprise a suitableamount of a pharmaceutically acceptable excipient so as to provide theform for proper administration. Pharmaceutical excipients can beliquids, such as water and oils, including those of petroleum, animal,vegetable, or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like. The pharmaceutical excipients can include,for example, saline, gum acacia, gelatin, starch paste, talc, keratin,colloidal silica, urea and the like. In addition, auxiliary,stabilizing, thickening, lubricating, and coloring agents can be used.In one embodiment, the pharmaceutically acceptable excipients aresterile when administered to a subject. Suitable pharmaceuticalexcipients also include starch, glucose, lactose, sucrose, gelatin,malt, rice, flour, chalk, silica gel, sodium stearate, glycerolmonostearate, talc, sodium chloride, dried skim milk, glycerol,propylene glycol, water, ethanol and the like. Any agent describedherein, if desired, can also comprise minor amounts of wetting oremulsifying agents, or pH buffering agents.

In one embodiment, the dosage form of the estrogen is an oralpreparation (liquid, tablet, capsule, caplet, or the like) which whenconsumed results in elevated serum estrogen levels. The oral preparationmay comprise conventional carriers including diluents, binders,time-release agents, lubricants, and disintegrants.

In other embodiments of the invention, the dosage form of the estrogenmay be provided in a topical preparation (lotion, cream, ointment,patch, or the like) for transdermal application.

Alternatively, the dosage form may be provided as a suppository or thelike for transvaginal or transrectal application.

However, in other embodiments, the dosage form may also allow forpreparations to be applied subcutaneously, intravenously,intramuscularly, or via the respiratory system.

In certain embodiments, the method includes the steps of administeringto the patient, on a continuous basis throughout two or more consecutivetreatment periods, a therapeutically effective amount of an estrogen;and administering to the patient, for only a portion of each treatmentperiod, a therapeutically effective amount of a progestogen.

The term “progestogen” (also known as “gestagen”) as used herein refersto any steroid hormone that binds to and activates a progesteronereceptor, or a precursor thereof. The term “progestogen” thus embracesnaturally occurring, synthetic, and semi-synthetic forms of progestogen,and biologically active, pharmaceutically acceptable salts and estersthereof.

In certain embodiments, the progestogen is selected from chlormadinoneacetate, cyproterone acetate, desogestrel, dienogest,5α-dihydroprogesterone, drospirenone (Yasmin®), ethinodiol acetate,ethynodiol diacetate, etonogestrel (Nexplanon®), gestodene,17-hydroxyprogesterone, levonorgestrel (Alesse®), medroxyprogesteroneacetate (17α-hydroxy-6α-methylprogesterone acetate; Provera®),megestrol, megestrol acetate(17α-acetoxy-6-dehydro-6-methylprogesterone), nestorone, nomegestrolacetate, norethindrone, norethindrone acetate (also known asnorethisterone acetate), norethynodrel (Enovid®), norgestimate,norgestrel, progesterone, tanaproget, trimegestone, or apharmaceutically acceptable salt of any of the foregoing, or anycombination thereof.

In certain embodiments, progestogen is a progestin. The term “progestin”as used herein refers to a synthetic progestogen as defined herein.Examples of progestins include desogestrel, dienogest, drospirenone(Yasmin®), ethinodiol acetate, etonogestrel (Nexplanon®), gestodene,levonorgestrel (Alesse®), medroxyprogesterone acetate (Provera®),nestorone, nomegestrol acetate, norethindrone, norethindrone acetate,norethynodrel (Enovid®), norgestimate, norgestrel, and trimegestone.

In certain embodiments, the progestogen is selected from progesterone,17-hydroxyprogesterone, 5α-dihydroprogesterone, norethindrone,norethindrone acetate (also known as norethisterone acetate),medroxyprogesterone acetate (17α-hydroxy-6α-methylprogesterone acetate),megestrol acetate (17α-acetoxy-6-dehydro-6-methylprogesterone),desogestrel, levonorgestrel, chlormadinone acetate, and cyproteroneacetate, pharmaceutically acceptable salts of any of the foregoing, andany combination thereof. In certain embodiments, progestogen is selectedfrom progesterone, 17-hydroxyprogesterone, 5α-dihydroprogesterone,norethindrone, norethindrone acetate (also known as norethisteroneacetate), desogestrel, levonorgestrel, chlormadinone acetate, andcyproterone acetate, pharmaceutically acceptable salts and esters of anyof the foregoing, and any combination thereof. In certain embodiments,progestogen is norethindrone or an ester thereof, or a pharmaceuticallyacceptable salt of an ester thereof, preferably norethindrone. Incertain embodiments, progestogen is progesterone or an ester thereof, ora pharmaceutically acceptable salt of an ester thereof.

In certain embodiments, the progestogen is administered in a dose equalor equivalent to about 70 μg to about 7 mg norethindrone daily, such asabout 100 μg to about 1 mg norethindrone daily, most preferably in adose equal or equivalent to about 0.7 mg norethindrone daily. In certainpreferred embodiments, the progestogen is norethindrone administered ina dose of 0.7 mg norethindrone daily.

In certain embodiments, the progestogen is formulated for oraladministration, e.g., in a dose equal or equivalent to about 70 μg toabout 7 mg norethindrone daily, such as about 100 μg to about 1 mgnorethindrone daily, most preferably in a dose equal or equivalent toabout 0.7 mg norethindrone daily. In certain preferred embodiments, theprogestogen is norethindrone formulated for oral administration in adose of 0.7 mg norethindrone daily.

In certain embodiments, the progestogen is orally administered in a doseequal or equivalent to about 70 μg to about 7 mg norethindrone daily,such as about 100 μg to about 1 mg norethindrone daily, most preferablyin a dose equal or equivalent to about 0.7 mg (i.e., 700 μg)norethindrone daily. In certain preferred embodiments, the progestogenis norethindrone orally administered in a dose of 0.7 mg (i.e., 700 μg)norethindrone daily.

A therapeutically effective dose of the progestogen included in thedosage form can be selected at least by considering the type ofprogestogen selected and the mode of administration. The dosage form mayinclude the progestogen in combination with other inert ingredients,including adjuvants and pharmaceutically acceptable carriers for thefacilitation of dosage to the patient as known to those skilled in thepharmaceutical arts. The dosage form may be any form suitable to causethe progestogen to enter into the tissues of the patient.

In one embodiment, the dosage form of the progestogen is an oralpreparation (liquid, tablet, capsule, caplet, or the like) which whenconsumed results in elevated serum progestogen levels. The oralpreparation may comprise conventional carriers including diluents,binders, time-release agents, lubricants, and disintegrants.

In other embodiments of the invention, the dosage form of theprogestogen may be provided in a topical preparation (lotion, cream,ointment, patch, or the like) for transdermal application.

Alternatively, the dosage form may be provided as a suppository or thelike for transvaginal or transrectal application.

In certain embodiments, the estrogen is administered to the subject on acontinuous basis throughout two or more consecutive treatment periods.In certain embodiments, a continuous basis means daily, i.e., onconsecutive days. For example, estrogen administered orally to a subjecton a daily basis throughout two or more consecutive treatment periods isdeemed to be estrogen administered to the subject on a continuous basisthroughout two or more consecutive treatment periods. Alternatively,estrogen administered transdermally to a subject on a daily basisthroughout two or more consecutive treatment periods is deemed to beestrogen administered to the subject on a continuous basis throughouttwo or more consecutive treatment periods.

As used herein, a “treatment period” refers to a period of time duringwhich a subject is receiving, on a continuous or daily basis, at leastone therapeutic agent administered for the purpose of treating MS in thesubject. In certain embodiments, each treatment period is at least 28consecutive days. In certain embodiments, each treatment period is atleast 56 consecutive days. In certain embodiments, each treatment periodis at least 84 consecutive days. In certain embodiments, each treatmentperiod is at least 112 consecutive days. In certain embodiments, eachtreatment period is at least 140 consecutive days. In certainembodiments, each treatment period is at least 168 consecutive days.

In certain embodiments, each treatment period is at least 4 consecutiveweeks. In certain embodiments, each treatment period is at least 8consecutive weeks. In certain embodiments, each treatment period is atleast 12 consecutive weeks. In certain embodiments, each treatmentperiod is at least 16 consecutive weeks. In certain embodiments, eachtreatment period is at least 20 consecutive weeks. In certainembodiments, each treatment period is at least 24 consecutive weeks.

In certain embodiments, each treatment period is at least one month. Incertain embodiments, each treatment period is at least two consecutivemonths. In certain embodiments, each treatment period is at least threeconsecutive months. In certain embodiments, each treatment period is atleast four consecutive months. In certain embodiments, each treatmentperiod is at least five consecutive months. In certain embodiments, eachtreatment period is at least six consecutive months.

In certain embodiments, the progestogen is administered to the subjectfor only a portion of each treatment period. As used herein, “for only aportion of each treatment period” refers generally to a period of timethat occurs during but is at least one day shorter than a treatmentperiod. In a preferred embodiment, the phrase “for only a portion ofeach treatment period” refers generally to a period of consecutive daysthat occurs during but is at least one day shorter than a treatmentperiod.

In certain embodiments, the portion of each treatment period is dailyfor all but at least 7 consecutive days of each treatment period. Forexample, if the treatment period is 28 days, in various embodiments theportion of such treatment period can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,1, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 consecutive days. Forconvenience, such portion can begin on day 1 of a treatment period, suchthat, for this example, the portion can encompass day 1 or days 1 to 2,1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to11, 1 to 12, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to19, 1 to 20, or 1 to 21.

In certain embodiments, the portion of each treatment period is dailyfor all but at least 14 consecutive days of each treatment period. Forexample, if the treatment period is 28 days, in various embodiments theportion of such treatment period can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, or 14 consecutive days. For convenience, such portion canbegin on day 1 of a treatment period, such that, for this example, theportion can encompass day 1 or days 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to 13, or 1 to14.

In certain embodiments, the portion of each treatment period is dailyfor up to 7 consecutive days of each treatment period. For example, ifthe treatment period is 28 days, in various embodiments the portion ofsuch treatment period can be 1, 2, 3, 4, 5, 6, or 7 consecutive days.For convenience, such portion can begin on day 1 of a treatment period,such that, for this example, the portion can encompass day 1 or days 1to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, or 1 to 7.

In certain embodiments, the portion of each treatment period is dailyfor up to 14 consecutive days of each treatment period. For example, ifthe treatment period is 28 days, in various embodiments the portion ofsuch treatment period can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,or 14 consecutive days. For convenience, such portion can begin on day 1of a treatment period, such that, for this example, the portion canencompass day 1 or days 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7,1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to 13, or 1 to 14.

In certain embodiments, the portion of each treatment period is dailyfor all but at least half of each treatment period. For example, if thetreatment period is 28 days, in various embodiments the portion of suchtreatment period can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14consecutive days. For convenience, such portion can begin on day 1 of atreatment period, such that, for this example, the portion can encompassday 1 or days 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1to 9, 1 to 10, 1 to 11, 1 to 12, 1 to 13, or to 14.

Preferably the progestogen is administered to the subject for only aportion of each treatment period. During the remainder of the treatmentperiod, in certain embodiments the subject can receive estrogen butneither progestogen nor a placebo in place of the progestogen.Alternatively, during the remainder of the treatment period, in certainembodiments the subject can receive both estrogen and a placebo in placeof the progestogen.

In certain embodiments, the method further includes the steps ofadministering orally to the patient, on a continuous basis for 84consecutive days (12 weeks), about 8 mg of estriol daily; andadministering orally to the patient, for 14 consecutive days (2 weeks)of the 84 consecutive days (12 weeks), about 0.7 mg of progestogendaily. In certain embodiments, the 14 consecutive days (2 weeks) are thefirst 14 consecutive days (2 weeks) of the 84 consecutive days (12weeks). That is, if the 84 consecutive days of estrogen administrationare deemed to start on day 1, the progestogen is administered on days 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14, and then stopped. Incertain embodiments, the patient may then continue to receive estrogenbut neither progestogen nor a placebo in place of the progestogen forthe remaining 70 days. In other embodiments, the method further includesthe step of administering to the patient a placebo in place of theprogestogen on each of the days the progestogen is not administered tothe patient. That is, the patient may then receive both estrogen and aplacebo in place of the progestogen for the remaining 70 days.

In certain embodiments, the method further includes the steps ofadministering orally to the patient, on a continuous basis for 84consecutive days (12 weeks), about 8 mg of estriol daily; andadministering orally to the patient, for 14 consecutive days (2 weeks)of the 84 consecutive days (12 weeks), about 0.7 mg of norethindronedaily. In certain embodiments, the 14 consecutive days (2 weeks) are thefirst 14 consecutive days (2 weeks) of the 84 consecutive days (12weeks). That is, if the 84 consecutive days of estrogen administrationare deemed to start on day 1, the norethindrone is administered on days1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14, and then stopped. Incertain embodiments, the patient may then continue to receive estrogenbut neither norethindrone nor a placebo in place of the norethindronefor the remaining 70 days. In other embodiments, the method furtherincludes the step of administering to the patient a placebo in place ofthe norethindrone on each of the days the norethindrone is notadministered to the patient. That is, the patient may then receive bothestrogen and a placebo in place of the norethindrone for the remaining70 days.

In certain embodiments, the method further comprises administering tothe patient an immunotherapeutic agent.

The term “immunotherapeutic agent” as used herein refers to a compoundwith an objectively measurable effect on at least one aspect of theimmune system or an immune response. In certain embodiments, theimmunotherapeutic agent is immunosuppressive, i.e., it exerts anobjectively measurable inhibitory effect on at least one aspect of theimmune system or an immune response. In certain embodiments, theimmunotherapeutic agent is anti-inflammatory. In certain embodiments,the immunotherapeutic agent is a small molecule (molecular weight lessthan or equal to about 1.5 kDa) pharmaceutical compound or composition.In certain embodiments, the immunotherapeutic agent is a biologicalcompound or composition, e.g., an antibody, peptide, nucleic acid, etc.

In certain embodiments, the immunotherapeutic agent is not an estrogen.In certain embodiments, the immunotherapeutic agent is not aprogestogen. Preferably, the immunotherapeutic agent is neither anestrogen nor a progestogen.

In certain embodiments, the immunotherapeutic agent is selected fromdimethyl fumarate (Tecfidera®; BG-12), fingolimod (Gilenya®), glatirameracetate (Copaxone®), for example “longer-lasting” 40 mg/ml or 20 mg/mlversions), interferon beta-1a (Avonex® or Rebif®), interferon beta-1b(Betaseron® or Extavia®)), mitoxantrone (Novantrone®), natalizumab(Tysabri®), and teriflunomide (Aubagio®), mycophenolate mofetil,paclitaxel, cyclosporine, corticosteroids (e.g., prednisone,methylprednisolone), azathioprine, cyclophosphamide, methotrexate,cladribine, 4-aminopyridine, and tizanidine. In certain embodiments, theimmunotherapeutic agent is selected from dimethyl fumarate (Tecfidera®;BG-12), fingolimod (Gilenya®), glatiramer acetate (Copaxone®),interferon beta-1a (Avonex® or Rebif®), interferon beta-1b (Betaseron®or Extavia®), mitoxantrone (Novantrone®), natalizumab (Tysabri®), andteriflunomide (Aubagio®).

In certain embodiments, the immunotherapeutic agent is dimethyl fumarate(Tecfidera®; BG-12). In certain embodiments, the immunotherapeutic agentis fingolimod (Gilenya®). In certain embodiments, the immunotherapeuticagent is glatiramer acetate (Copaxone®). In certain embodiments, theimmunotherapeutic agent is interferon beta-1a (Avonex® or Rebif®). Incertain embodiments, the immunotherapeutic agent is interferon beta-1b(Betaseron® or Extavia®). In certain embodiments, the immunotherapeuticagent is mitoxantrone (Novantrone®). In certain embodiments, theimmunotherapeutic agent is natalizumab (Tysabri®). In certainembodiments, the immunotherapeutic agent is teriflunomide (Aubagio®).

In certain embodiments, the immunotherapeutic agent is an agent thatreduces the activity of LINGO-1, e.g., anti-LINGO-1 antibody (BIIB033,Biogen-Idec).

In certain embodiments, treatment with the immunotherapeutic agent isinitiated at the same time or about the same time as initiation oftreatment with the estrogen.

In certain embodiments, the immunotherapeutic agent is selected frominterferon-beta-1a, interferon-beta-1b, glatiramer acetate, natalizumab,mitoxantrone, fingolimod, teriflunomide, dimethyl fumarate,mycophenolate mofetil, paclitaxel, cyclosporine, corticosteroids,azathioprine, cyclophosphamide, methotrexate, cladribine,4-aminopyridine, and tizanidine.

In certain embodiments, the immunotherapeutic agent is selected frominterferon-beta 1a, interferon-beta 1b, glatiramer acetate, natalizumab,mitoxantrone, fingolimod, teriflunomide, and dimethyl fumarate.

In certain embodiments, the immunotherapeutic agent is glatirameracetate.

An aspect of the invention is a method of treating a neurodegenerativedisease, comprising

administering to a subject in need thereof a treatment regimen;

assessing a change in total (whole) brain gray matter volume of at leastone brain region of the subject over a period of time; and

changing the treatment regimen if the total (whole) brain gray mattervolume decreases by at least about 0.3 percent between a firstassessment and a second assessment. In certain embodiments, the secondassessment occurs at least about 6 months after the first assessment,such as about six months after the first assessment. In otherembodiments, the second assessment occurs about one year after the firstassessment, and the treatment regimen is changed if the total (whole)brain gray matter volume decreases by at least about 0.6 percent betweenthe first assessment and the second assessment. In yet otherembodiments, the second assessment occurs about two years after thefirst assessment, and the treatment regimen is changed if the total(whole) brain gray matter volume decreases by at least about 1.0 percentbetween the first assessment and the second assessment.

The term “subject” as used herein refers to a living mammal and may beinterchangeably used with the term “patient”. In certain embodiments,the subject is a human. Preferably, the human subject is female, such asa woman. In certain embodiments, the subject is a premenopausal orperimenopausal woman. In certain embodiments, the subject is apremenopausal woman. In certain embodiments, the subject is aperimenopausal woman. In certain embodiments, the subject is apostmenopausal woman.

In certain embodiments, a subject for a method as described herein hasone or more of: experienced a first clinical episode, MRI featuresconsistent with multiple sclerosis, an inadequate response to analternate MS therapy (e.g., one or more of dimethyl fumarate(Tecfidera®; BG-12), fingolimod (Gilenya®), glatiramer acetate(Copaxone®), interferon beta-1a (e.g., Avonex® and Rebif®), interferonbeta-1b (e.g., Betaseron® and Extavia®), mitoxantrone (Novantrone®),natalizumab (Tysabri®), teriflunomide (Aubagio®) and anti-LINGO-1antibody (BIIB033, Biogen-Idec), and an inability to tolerate analternate MS therapy (e.g., one or more of dimethyl fumarate(Tecfidera®; BG-12), fingolimod (Gilenya®), glatiramer acetate(Copaxone®), interferon beta-1a (Avonex® and Rebif®), interferon beta-1b(e.g., Betaseron® and Extavia®), mitoxantrone (Novantrone®), natalizumab(Tysabri®), teriflunomide (Aubagio®), and anti-LINGO-1 antibody(BIIB033, Biogen-Idec)).

In certain embodiments, the neurodegenerative disease is Alzheimer'sdisease, Parkinson's disease, multiple sclerosis, stroke, amyotrophiclateral sclerosis, cerebellar ataxia, frontotemporal dementia, priondisease, Huntington's disease, cerebral ischemia, cerebral dementiasyndrome, infection-induced neurodegeneration disorders (e.g.,AIDS-encephalopathy, Creutzfeld-Jakob disease, encephalopathies inducedby rubiola and herpes viruses and borrelioses), metabolic-toxicneurodegenerative disorders (such as hepatic-, alcoholic-, hypoxic-,hypo- or hyperglycemically-induced encephalopathies), encephalopathiesinduced by solvents or pharmaceuticals, trauma-induced brain damage, orspinal cord injury.

In certain embodiments, the neurodegenerative disease is Alzheimer'sdisease, Parkinson's disease, multiple sclerosis, stroke, amyotrophiclateral sclerosis, cerebellar ataxia, frontotemporal dementia, priondisease, Huntington's disease, cerebral ischemia, cerebral dementiasyndrome, infection-induced neurodegeneration disorders (e.g.,AIDS-encephalopathy, Creutzfeld-Jakob disease, encephalopathies inducedby rubiola and herpes viruses and borrelioses), metabolic-toxicneurodegenerative disorders (such as hepatic-, alcoholic-, hypoxic-,hypo- or hyperglycemically-induced encephalopathies), encephalopathiesinduced by solvents or pharmaceuticals, or trauma-induced brain damage.

In certain embodiments, the neurodegenerative disease is multiplesclerosis.

In certain embodiments, the neurodegenerative disease is clinicallyisolated syndrome (CIS).

In certain embodiments, the neurodegenerative disease isrelapsing-remitting multiple sclerosis.

In certain embodiments, the neurodegenerative disease issecondary-progressive multiple sclerosis.

In certain embodiments, the neurodegenerative disease isprimary-progressive multiple sclerosis.

In certain embodiments, the neurodegenerative disease isprogressive-relapsing multiple sclerosis.

As used herein, in certain embodiments, “total (whole) brain graymatter” refers to the combined volume of all intracranial gray matter,i.e., rostral to the cervical spinal cord. As used herein, in certainembodiments, “total (whole) brain gray matter” refers to the combinedvolume of gray matter of cerebral cortex, cerebellum, thalamus, caudatenucleus, and putamen.

In certain embodiments, the assessing comprises performing brain MRI.

In certain embodiments, the assessing consists of performing brain MRI.

In certain embodiments, the second assessment is performed about 2, 3,4, 5, 6, 7, 8, 9, 10, or 11 months after the first assessment.

In certain embodiments, the second assessment is performed about 3months after the first assessment.

In certain embodiments, the second assessment is performed about 4months after the first assessment.

In certain embodiments, the second assessment is performed about 6months after the first assessment.

In certain embodiments, the second assessment is performed at leastabout 6 months, at least about one year, at least about 18 months, or atleast about two years after the first assessment.

In certain embodiments, the second assessment is performed at leastabout one year after the first assessment. For example, in variousembodiments, the second assessment is performed about 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, or 24 or more months after the firstassessment. In various embodiments, the second assessment is performedabout 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more years after the firstassessment.

In certain embodiments, the second assessment is performed about oneyear after the first assessment.

In certain embodiments, the second assessment is performed about twoyears after the first assessment.

In certain embodiments, the first assessment is performed before, at thesame time as, or at about the same time as initiating the treatmentregimen. In certain embodiments, the first assessment is performed afterthe treatment regimen is initiated.

The method includes the step of changing the treatment regimen if thetotal (whole) brain gray matter volume decreases by at least about 0.3percent between a first assessment and a second assessment. In certainembodiments, the total (whole) brain gray matter volume decreases by0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, or greater than 1.0 percent betweenthe first assessment and the second assessment.

As used herein, the phrase “treatment regimen” means a repeated orstructured intervention made over a period of time with the intention oftreating at least one manifestation of a disease or condition of asubject. A treatment regimen typically includes administration of atleast one agent useful or believed to be useful in the treatment of thedisease or condition. In certain embodiments, a treatment regimenincludes administration of a single agent useful or believed to beuseful in the treatment of the disease or condition. In certainembodiments, a treatment regimen includes administration of two agentsuseful or believed to be useful in the treatment of the disease orcondition. In certain embodiments, a treatment regimen includesadministration of three agents useful or believed to be useful in thetreatment of the disease or condition.

As used herein, the phrase “changing the treatment regimen” meanschanging the amount or inclusion of at least one agent in the treatmentregimen. For example, an agent can be increased, decreased, stopped, orreplaced by another agent. In certain embodiments, a further agent canbe added to the treatment regimen. For example, in certain embodiments,changing the treatment regimen involves adding estrogen, with or withoutprogestogen, to the treatment regimen.

In certain embodiments, the treatment regimen does not compriseestrogen. In certain such embodiments, changing the treatment regimencomprises adding administration of estrogen to the treatment regimen.For example, if the treatment regimen comprises administering animmunotherapeutic agent but not estrogen, changing the treatment regimenmay comprise adding administration of estrogen (e.g., according to anyof the various protocols described herein) to the treatment regimen. Incertain embodiments, the estrogen added to the treatment regimen isselected from estriol (E3), estradiol (E2), estrone (E1), an esterthereof, a pharmaceutically acceptable salt of an ester thereof, and anycombination thereof, such as estriol (E3) or an ester thereof, or apharmaceutically acceptable salt of an ester thereof, preferablyestriol. In certain embodiments, the estrogen is administeredcontinuously on a daily basis. In certain embodiments, the subject isfemale.

In certain embodiments, the treatment regimen consists of administeringto the subject an immunotherapeutic agent. In certain embodiments, theimmunotherapeutic agent is not an estrogen. In certain embodiments, theimmunotherapeutic agent is not a progestogen. Preferably, theimmunotherapeutic agent is neither an estrogen nor a progestogen. Incertain embodiments, the immunotherapeutic agent is selected fromdimethyl fumarate (Tecfidera®; BG-12), fingolimod (Gilenya®), glatirameracetate (Copaxone®, for example “longer-lasting” 40 mg/ml or 20 mg/mlversions), interferon beta-1a (Avonex® or Rebif®), interferon beta-1b(Betaseron® or Extavia®), mitoxantrone (Novantrone®), natalizumab(Tysabri®), and teriflunomide (Aubagio®), mycophenolate mofetil,paclitaxel, cyclosporine, corticosteroids (e.g., prednisone,methylprednisolone), azathioprine, cyclophosphamide, methotrexate,cladribine, 4-aminopyridine, and tizanidine. In certain embodiments, theimmunotherapeutic agent is selected from dimethyl fumarate (Tecfidera®;BG-12), fingolimod (Gilenya®), glatiramer acetate (Copaxone®),interferon beta-1a (Avonex® or Rebif®), interferon beta-1b (Betaseron®or Extavia®), mitoxantrone (Novantrone®), natalizumab (Tysabri®), andteriflunomide (Aubagio®). In certain embodiments, the immunotherapeuticagent is dimethyl fumarate (Tecfidera®; BG-12). In certain embodiments,the immunotherapeutic agent is fingolimod (Gilenya®). In certainembodiments, the immunotherapeutic agent is glatiramer acetate(Copaxone®). In certain embodiments, the immunotherapeutic agent isinterferon beta-1a (Avonex® or Rebif®). In certain embodiments, theimmunotherapeutic agent is interferon beta-1 b (Betaseron®) orExtavia®). In certain embodiments, the immunotherapeutic agent ismitoxantrone (Novantrone®). In certain embodiments, theimmunotherapeutic agent is natalizumab (Tysabri®). In certainembodiments, the immunotherapeutic agent is teriflunomide (Aubagio®). Incertain embodiments, the immunotherapeutic agent is an agent thatreduces the activity of LINGO-1, e.g., anti-LINGO-1 antibody (BIIB033,Biogen-Idec).

In certain embodiments, the treatment regimen comprises administering tothe subject an estrogen. In certain embodiments, the estrogen isselected from estriol (E3), estradiol (E2), estrone (E1), an esterthereof, a pharmaceutically acceptable salt of an ester thereof, and anycombination thereof.

In certain embodiments, estrogen is estriol (E3) or an ester thereof, ora pharmaceutically acceptable salt of an ester thereof. In certainembodiments, the estrogen is estriol.

In certain embodiments, the estrogen is administered continuously on adaily basis.

In certain embodiments, the subject is female.

In certain embodiments, the estrogen is administered orally in a doseequal or equivalent to about 200 μg to about 20 mg of estriol daily.

In certain embodiments, the estrogen is administered orally in a doseequal or equivalent to about 1 mg to about 10 mg of estriol daily.

In certain embodiments, the estrogen is administered orally in a doseequal or equivalent to about 8 mg of estriol daily.

In certain embodiments, adding administration of estrogen to thetreatment regimen comprises administering to the subject, on acontinuous basis throughout one or more consecutive treatment periods, atherapeutically effective amount of an estrogen; and

administering to the subject, for only a portion of each treatmentperiod, a therapeutically effective amount of a progestogen.

In certain embodiments, the treatment regimen continues for at least twotreatment periods.

In certain embodiments, the progestogen is selected from chlormadinoneacetate, cyproterone acetate, desogestrel, dienogest,5α-dihydroprogesterone, drospirenone, ethinodiol acetate, ethynodioldiacetate, etonogestrel, gestodene, 17-hydroxyprogesterone,levonorgestrel, medroxyprogesterone acetate(17α-hydroxy-6α-methylprogesterone acetate), megestrol, megestrolacetate (7α-acetoxy-6-dehydro-6-methylprogesterone), nestorone,nomegestrol acetate, norethindrone, norethindrone acetate,norethynodrel, norgestimate, norgestrel, progesterone, tanaproget,trimegestone, pharmaceutically acceptable salts of any of the foregoing,and any combination thereof.

In certain embodiments, the progestogen is progesterone.

In certain embodiments, the progestogen is norethindrone.

In certain embodiments, each treatment period is at least 28 consecutivedays.

In certain embodiments, each treatment period is at least 56 consecutivedays.

In certain embodiments, each treatment period is at least 84 consecutivedays.

In certain embodiments, each treatment period is at least 112consecutive days.

In certain embodiments, each treatment period is at least 4 consecutiveweeks.

In certain embodiments, each treatment period is at least 8 consecutiveweeks.

In certain embodiments, each treatment period is at least 12 consecutiveweeks.

In certain embodiments, each treatment period is at least 16 consecutiveweeks.

In certain embodiments, each treatment period is at least one month.

In certain embodiments, each treatment period is at least twoconsecutive months.

In certain embodiments, each treatment period is at least threeconsecutive months.

In certain embodiments, each treatment period is at least fourconsecutive months.

In certain embodiments, the portion of each treatment period is dailyfor all but at least 7 consecutive days of each treatment period.

In certain embodiments, the portion of each treatment period is dailyfor all but at least 14 consecutive days of each treatment period.

In certain embodiments, the portion of each treatment period is dailyfor up to 7 consecutive days of each treatment period.

In certain embodiments, the portion of each treatment period is dailyfor up to 14 consecutive days of each treatment period.

In certain embodiments, the portion of each treatment period is dailyfor all but at least half of each treatment period.

In certain embodiments, the progestogen is administered orally in a doseequal or equivalent to about 70 μg to about 7 mg of norethindrone daily.

In certain embodiments, the progestogen is administered orally in a doseequal or equivalent to about 100 μg to about 1 mg of norethindronedaily.

In certain embodiments, the progestogen is administered orally in a doseequal or equivalent to about 700 μg of norethindrone daily.

In certain embodiments, the estrogen is administered orally in a doseequal or equivalent to about 200 μg to about 20 mg of estriol daily.

In certain embodiments, the estrogen is administered orally in a doseequal or equivalent to about 1 mg to about 10 mg of estriol daily.

In certain embodiments, the estrogen is administered orally in a doseequal or equivalent to about 8 mg of estriol daily.

In certain embodiments, the treatment regimen further comprisesadministering to the subject an immunotherapeutic agent.

In certain embodiments, the immunotherapeutic agent isimmunosuppressive. In certain embodiments, the immunotherapeutic agentis anti-inflammatory. In certain embodiments, the immunotherapeuticagent is a small molecule (molecular weight less than or equal to about1.5 kDa) pharmaceutical compound or composition. In certain embodiments,the immunotherapeutic agent is a biological compound or composition,e.g., an antibody, peptide, nucleic acid, etc.

In certain embodiments, the immunotherapeutic agent is not an estrogen.In certain embodiments, the immunotherapeutic agent is not aprogestogen. Preferably, the immunotherapeutic agent is neither anestrogen nor a progestogen.

In certain embodiments, the immunotherapeutic agent is selected fromdimethyl fumarate (Tecfidera®; BG-12), fingolimod (Gilenya®), glatirameracetate (Copaxone®, for example “longer-lasting” 40 mg/ml or 20 mg/mlversions), interferon beta-1a (Avonex® or Rebif®), interferon beta-1b(Betaseron® or Extavia®), mitoxantrone (Novantrone®), natalizumab(Tysabri®), and teriflunomide (Aubagio®), mycophenolate mofetil,paclitaxel, cyclosporine, corticosteroids (e.g., prednisone,methylprednisolone), azathioprine, cyclophosphamide, methotrexate,cladribine, 4-aminopyridine, and tizanidine. In certain embodiments, theimmunotherapeutic agent is selected from dimethyl fumarate (Tecfidera®;BG-12), fingolimod (Gilenya®), glatiramer acetate (Copaxone®),interferon beta-1a (Avonex® or Rebif®), interferon beta-1b (Betaseron®or Extavia®), mitoxantrone (Novantrone®), natalizumab (Tysabri®), andteriflunomide (Aubagio®).

In certain embodiments, the immunotherapeutic agent is dimethyl fumarate(Tecfidera®; BG-12). In certain embodiments, the immunotherapeutic agentis fingolimod (Gilenya®). In certain embodiments, the immunotherapeuticagent is glatiramer acetate (Copaxone®)). In certain embodiments, theimmunotherapeutic agent is interferon beta-1a (Avonex® or Rebif®). Incertain embodiments, the immunotherapeutic agent is interferon beta-1b(Betaseron® or Extavia®). In certain embodiments, the immunotherapeuticagent is mitoxantrone (Novantrone®). In certain embodiments, theimmunotherapeutic agent is natalizumab (Tysabri®). In certainembodiments, the immunotherapeutic agent is teriflunomide (Aubagio®)).

In certain embodiments, the immunotherapeutic agent is an agent thatreduces the activity of LINGO-1, e.g., anti-LINGO-1 antibody (BIIB033,Biogen-Idec).

In certain embodiments, treatment with the immunotherapeutic agent isinitiated at the same time or about the same time as initiation oftreatment with the estrogen.

In certain embodiments, the immunotherapeutic agent is selected frominterferon-beta 1a, interferon-beta 1b, glatiramer acetate, natalizumab,mitoxantrone, fingolimod, teriflunomide, dimethyl fumarate,mycophenolate mofetil, paclitaxel, cyclosporine, corticosteroids,azathioprine, cyclophosphamide, methotrexate, cladribine,4-aminopyridine, and tizanidine.

In certain embodiments, the immunotherapeutic agent is selected frominterferon-beta 1a, interferon-beta 1b, glatiramer acetate, natalizumab,mitoxantrone, fingolimod, teriflunomide, and dimethyl fumarate.

In certain embodiments, the immunotherapeutic agent is glatirameracetate.

An aspect of the invention is a method of treating a neurodegenerativedisease, comprising

administering to a human subject in need thereof a treatment regimen;

assessing a change in gray matter volume of at least two brain regionsof the subject over a period of time; and

changing the treatment regimen if the gray matter volume of the at leasttwo brain regions decreases by at least about 0.3 percent between afirst assessment and a second assessment. In certain embodiments, thesecond assessment occurs at least about 6 months after the firstassessment, such as about six months after the first assessment. Inother embodiments, the second assessment occurs about one year after thefirst assessment, and the treatment regimen is changed if the graymatter volume of the at least two brain regions decreases by at leastabout 0.6 percent between the first assessment and the secondassessment. In yet other embodiments, the second assessment occurs abouttwo years after the first assessment, and the treatment regimen ischanged if the gray matter volume of the at least two brain regionsdecreases by at least about 1.0 percent between the first assessment andthe second assessment.

In certain embodiments, the neurodegenerative disease is Alzheimer'sdisease, Parkinson's disease, multiple sclerosis, stroke, amyotrophiclateral sclerosis, cerebellar ataxia, frontotemporal dementia, priondisease, Huntington's disease, cerebral ischemia, cerebral dementiasyndrome, infection-induced neurodegeneration disorders (e.g.,AIDS-encephalopathy, Creutzfeld-Jakob disease, encephalopathies inducedby rubiola and herpes viruses and borrelioses), metabolic-toxicneurodegenerative disorders (such as hepatic-, alcoholic-, hypoxic-,hypo- or hyperglycemically-induced encephalopathies), encephalopathiesinduced by solvents or pharmaceuticals, trauma-induced brain damage, orspinal cord injury.

In certain embodiments, the neurodegenerative disease is Alzheimer'sdisease, Parkinson's disease, multiple sclerosis, stroke, amyotrophiclateral sclerosis, cerebellar ataxia, frontotemporal dementia, priondisease, Huntington's disease, cerebral ischemia, cerebral dementiasyndrome, infection-induced neurodegeneration disorders (e.g.,AIDS-encephalopathy, Creutzfeld-Jakob disease, encephalopathies inducedby rubiola and herpes viruses and borrelioses), metabolic-toxicneurodegenerative disorders (such as hepatic-, alcoholic-, hypoxic-,hypo- or hyperglycemically-induced encephalopathies), encephalopathiesinduced by solvents or pharmaceuticals, or trauma-induced brain damage.

In certain embodiments, the neurodegenerative disease is multiplesclerosis.

In certain embodiments, the neurodegenerative disease is clinicallyisolated syndrome (CIS).

In certain embodiments, the neurodegenerative disease isrelapsing-remitting multiple sclerosis.

In certain embodiments, the neurodegenerative disease issecondary-progressive multiple sclerosis.

In certain embodiments, the neurodegenerative disease isprimary-progressive multiple sclerosis.

In certain embodiments, the neurodegenerative disease isprogressive-relapsing multiple sclerosis.

In certain embodiments, the treatments described herein involve one ormore of preventing disease progression, slowing of disease progression,reducing the number of disease relapses or clinical exacerbations,slowing memory loss, and slowing the accumulation of physicaldisability.

In certain embodiments, the assessing comprises performing brain MRI.

In certain embodiments, the assessing consists of performing brain MRI.

In certain embodiments, the at least two brain regions are selected fromcerebral cortex, cerebellum, thalamus, caudate nucleus, putamen, and anycombination thereof.

In certain embodiments, one of the at least two brain regions comprisescerebral cortex.

In certain embodiments, the second assessment is performed about 2, 3,4, 5, 6, 7, 8, 9, 10, or 11 months after the first assessment.

In certain embodiments, the second assessment is performed about 3months after the first assessment.

In certain embodiments, the second assessment is performed about 4months after the first assessment.

In certain embodiments, the second assessment is performed about 6months after the first assessment.

In certain embodiments, the second assessment is performed at leastabout 6 months, at least about one year, at least about 18 months, or atleast about two years after the first assessment.

In certain embodiments, the second assessment is performed at leastabout one year after the first assessment. For example, in variousembodiments, the second assessment is performed about 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, or 24 or more months after the firstassessment. In various embodiments, the second assessment is performedabout 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more years after the firstassessment.

In certain embodiments, the second assessment is performed about oneyear after the first assessment.

In certain embodiments, the second assessment is performed about twoyears after the first assessment.

In certain embodiments, the first assessment is performed before, at thesame time as, or at about the same time as initiating the treatmentregimen. In certain embodiments, the first assessment is performed afterthe treatment regimen is initiated.

The method includes the step of changing the treatment regimen if thegray matter volume decreases by at least about 4 mL between a firstassessment and a second assessment. In certain embodiments, the graymatter volume decreases by 4, 5, 6, 7, 8, 9, 10, or greater than 10 mLbetween the first assessment and the second assessment.

In certain embodiments, the treatment regimen does not compriseestrogen, and changing the treatment regimen comprises addingadministration of estrogen (e.g., according to any of the variousprotocols described herein) to the treatment regimen. For example, ifthe treatment regimen comprises administering an immunotherapeutic agentbut not estrogen, changing the treatment regimen may comprise addingadministration of estrogen to the treatment regimen. In certainembodiments, the estrogen added to the treatment regimen is selectedfrom estriol (E3), estradiol (E2), estrone (E1), an ester thereof, apharmaceutically acceptable salt of an ester thereof, and anycombination thereof, such as estriol (E3) or an ester thereof, or apharmaceutically acceptable salt of an ester thereof, preferablyestriol. In certain embodiments, the estrogen is administeredcontinuously on a daily basis. In certain embodiments, the subject isfemale.

In certain embodiments, the treatment regimen consists of administeringto the subject an immunotherapeutic agent. In certain embodiments, theimmunotherapeutic agent is not an estrogen. In certain embodiments, theimmunotherapeutic agent is not a progestogen. Preferably, theimmunotherapeutic agent is neither an estrogen nor a progestogen. Incertain embodiments, the immunotherapeutic agent is selected fromdimethyl fumarate (Tecfidera®; BG-12), fingolimod (Gilenya®), glatirameracetate (Copaxone®, for example “longer-lasting” 40 mg/ml or 20 mg/mlversions), interferon beta-1a (e.g., Avonex® or Rebif®), interferonbeta-1b (e.g., Betaseron® or Extavia®), mitoxantrone (Novantrone®),natalizumab (Tysabri®), and teriflunomide (Aubagio®), mycophenolatemofetil, paclitaxel, cyclosporine, corticosteroids (e.g., prednisone,methylprednisolone), azathioprine, cyclophosphamide, methotrexate,cladribine, 4-aminopyridine, and tizanidine. In certain embodiments, theimmunotherapeutic agent is selected from dimethyl fumarate (Tecfidera®;BG-12), fingolimod (Gilenya®), glatiramer acetate (Copaxone®),interferon beta-1a (Avonex® or Rebif®), interferon beta-1b (e.g.,Betaseron® or Extavia®), mitoxantrone (Novantrone®), natalizumab(Tysabri®), and teriflunomide (Aubagio®). In certain embodiments, theimmunotherapeutic agent is dimethyl fumarate (Tecfidera®; BG-12). Incertain embodiments, the immunotherapeutic agent is fingolimod(Gilenya®). In certain embodiments, the immunotherapeutic agent isglatiramer acetate (Copaxone®). In certain embodiments, theimmunotherapeutic agent is interferon beta-1a (Avonex® or Rebif®). Incertain embodiments, the immunotherapeutic agent is interferon beta-1b(e.g., Betaseron® or Extavia®). In certain embodiments, theimmunotherapeutic agent is mitoxantrone (Novantrone®). In certainembodiments, the immunotherapeutic agent is natalizumab (Tysabri®). Incertain embodiments, the immunotherapeutic agent is teriflunomide(Aubagio®). In certain embodiments, the immunotherapeutic agent is anagent that reduces the activity of LINGO-1, e.g., anti-LINGO-1 antibody(BIIB033, Biogen-Idec).

In certain embodiments, the treatment regimen comprises administering tothe subject an estrogen. In certain embodiments, the estrogen isselected from estriol (E3), estradiol (E2), estrone (E1), an esterthereof, a pharmaceutically acceptable salt of an ester thereof, and anycombination thereof.

In certain embodiments, estrogen is estriol (E3) or an ester thereof, ora pharmaceutically acceptable salt of an ester thereof. In certainembodiments, the estrogen is estriol.

In certain embodiments, the estrogen is administered continuously on adaily basis.

In certain embodiments, the subject is female.

In certain embodiments, the estrogen is administered orally in a doseequal or equivalent to about 200 μg to about 20 mg of estriol daily.

In certain embodiments, the estrogen is administered orally in a doseequal or equivalent to about 1 mg to about 10 mg of estriol daily.

In certain embodiments, the estrogen is administered orally in a doseequal or equivalent to about 8 mg of estriol daily.

In certain embodiments, adding administration of estrogen to thetreatment regimen comprises administering to the subject, on acontinuous basis throughout one or more consecutive treatment periods, atherapeutically effective amount of an estrogen; and

administering to the subject, for only a portion of each treatmentperiod, a therapeutically effective amount of a progestogen.

In certain embodiments, the treatment regimen continues for at least twotreatment periods.

In certain embodiments, the progestogen is selected from chlormadinoneacetate, cyproterone acetate, desogestrel, dienogest,5α-dihydroprogesterone, drospirenone, ethinodiol acetate, ethynodioldiacetate, etonogestrel, gestodene, 17-hydroxyprogesterone,levonorgestrel, medroxyprogesterone acetate(17α-hydroxy-6α-methylprogesterone acetate), megestrol, megestrolacetate (17α-acetoxy-6-dehydro-6-methylprogesterone), nestorone,nomegestrol acetate, norethindrone, norethindrone acetate,norethynodrel, norgestimate, norgestrel, progesterone, tanaproget,trimegestone, pharmaceutically acceptable salts of any of the foregoing,and any combination thereof.

In certain embodiments, the progestogen is progesterone.

In certain embodiments, the progestogen is norethindrone.

In certain embodiments, each treatment period is at least 28 consecutivedays.

In certain embodiments, each treatment period is at least 56 consecutivedays.

In certain embodiments, each treatment period is at least 84 consecutivedays.

In certain embodiments, each treatment period is at least 112consecutive days.

In certain embodiments, each treatment period is at least 4 consecutiveweeks.

In certain embodiments, each treatment period is at least 8 consecutiveweeks.

In certain embodiments, each treatment period is at least 12 consecutiveweeks.

In certain embodiments, each treatment period is at least 16 consecutiveweeks.

In certain embodiments, each treatment period is at least one month.

In certain embodiments, each treatment period is at least twoconsecutive months.

In certain embodiments, each treatment period is at least threeconsecutive months.

In certain embodiments, each treatment period is at least fourconsecutive months.

In certain embodiments, the portion of each treatment period is dailyfor all but at least 7 consecutive days of each treatment period.

In certain embodiments, the portion of each treatment period is dailyfor all but at least 14 consecutive days of each treatment period.

In certain embodiments, the portion of each treatment period is dailyfor up to 7 consecutive days of each treatment period.

In certain embodiments, the portion of each treatment period is dailyfor up to 14 consecutive days of each treatment period.

In certain embodiments, the portion of each treatment period is dailyfor all but at least half of each treatment period.

In certain embodiments, the progestogen is administered orally in a doseequal or equivalent to about 70 μg to about 7 mg of norethindrone daily.

In certain embodiments, the progestogen is administered orally in a doseequal or equivalent to about 100 μg to about 1 mg of norethindronedaily.

In certain embodiments, the progestogen is administered orally in a doseequal or equivalent to about 700 μg of norethindrone daily.

In certain embodiments, the estrogen is administered orally in a doseequal or equivalent to about 200 μg to about 20 mg of estriol daily.

In certain embodiments, the estrogen is administered orally in a doseequal or equivalent to about 1 mg to about 10 mg of estriol daily.

In certain embodiments, the estrogen is administered orally in a doseequal or equivalent to about 8 mg of estriol daily.

In certain embodiments, the treatment regimen further comprisesadministering to the subject an immunotherapeutic agent.

In certain embodiments, the immunotherapeutic agent isimmunosuppressive. In certain embodiments, the immunotherapeutic agentis anti-inflammatory. In certain embodiments, the immunotherapeuticagent is a small molecule (molecular weight less than or equal to about1.5 kDa) pharmaceutical compound or composition. In certain embodiments,the immunotherapeutic agent is a biological compound or composition,e.g., an antibody, peptide, nucleic acid, etc.

In certain embodiments, the immunotherapeutic agent is not an estrogen.In certain embodiments, the immunotherapeutic agent is not aprogestogen. Preferably, the immunotherapeutic agent is neither anestrogen nor a progestogen.

In certain embodiments, the immunotherapeutic agent is selected fromdimethyl fumarate (Tecfidera®; BG-12), fingolimod (Gilenya®), glatirameracetate (Copaxone®, for example “longer-lasting” 40 mg/ml or 20 mg/mlversions), interferon beta-1a (Avonex® or Rebif®), interferon beta-1b(Betaseron® or Extavia®), mitoxantrone (Novantrone®), natalizumab(Tysabri®), and teriflunomide (Aubagio®), mycophenolate mofetil,paclitaxel, cyclosporine, corticosteroids (e.g., prednisone,methylprednisolone), azathioprine, cyclophosphamide, methotrexate,cladribine, 4-aminopyridine, and tizanidine. In certain embodiments, theimmunotherapeutic agent is selected from dimethyl fumarate (Tecfidera®;BG-12), fingolimod (Gilenya®), glatiramer acetate (Copaxone®),interferon beta-1a (Avonex® or Rebif®), interferon beta-1b (Betaseron®or Extavia®), mitoxantrone (Novantrone®), natalizumab (Tysabri®), andteriflunomide (Aubagio®).

In certain embodiments, the immunotherapeutic agent is dimethyl fumarate(Tecfidera®; BG-12). In certain embodiments, the immunotherapeutic agentis fingolimod (Gilenya®). In certain embodiments, the immunotherapeuticagent is glatiramer acetate (Copaxone®)). In certain embodiments, theimmunotherapeutic agent is interferon beta-1a (Avonex® or Rebif®). Incertain embodiments, the immunotherapeutic agent is interferon beta-1b(Betaseron® or Extavia®). In certain embodiments, the immunotherapeuticagent is mitoxantrone (Novantrone®). In certain embodiments, theimmunotherapeutic agent is natalizumab (Tysabri®). In certainembodiments, the immunotherapeutic agent is teriflunomide (Aubagio®).

In certain embodiments, the immunotherapeutic agent is an agent thatreduces the activity of LINGO-1, e.g., anti-LINGO-1 antibody (BIIB033,Biogen-Idec).

In certain embodiments, treatment with the immunotherapeutic agent isinitiated at the same time or about the same time as initiation oftreatment with the estrogen.

The various methods disclosed herein can be methods for improvingwalking, vision, balance, cognition, memory, or other symptoms in asubject, such as a subject with multiple sclerosis, and/or methods forimproving multiple sclerosis functional composite (MSFC), EDSS, or MSSSscores in a subject, such as a subject with multiple sclerosis. Thus, incertain embodiments, the methods of treatment disclosed herein includemethods for improving disability in a patient, whereby the patient'sdisability score (as measured by either of these tests or anothersuitable test) after two years of therapy is at least about 10%, atleast about 25%, at least about 40%, at least about 50%, or even atleast about 60% higher relative to a control patient not receiving theestrogen/progestogen therapy (but otherwise receiving the same treatmentas the estrogen-treated patient).

In various embodiments, the present methods treat or prevent one or moresymptoms of a neurodegenerative disease (e.g. MS) including, by way ofillustration: optic neuritis, diplopia, nystagmus, ocular dysmetria,internuclear opthalmoplegia, movement and sound phosphenes, afferentpupillary defect, paresis, monoparesis, paraparesis, hemiparesis,quadraparesis, plegia, paraplegia, hemiplegia, tetraplegia,quadriplegia, spasticity, dysarthria, muscle atrophy, spasms, cramps,hypotonia, clonus, myoclonus, myokymia, restless leg syndrome, footdrop,dysfunctional reflexes, paraesthesia, anaesthesia, neuralgia,neuropathic and neurogenic pain, L'Hermitte's sign, proprioceptivedysfunction, trigeminal neuralgia, ataxia, intention tremor, dysmetria,vestibular ataxia, vertigo, speech ataxia, dystonia, dysdiadochokinesia,frequent micturation, bladder spasticity, flaccid bladder,detrusor-sphincter dyssynergia, erectile dysfunction, anorgamy,frigidity, constipation, fecal urgency, fecal incontinence, depression,cognitive dysfunction, memory loss, dementia, mood swings, emotionallability, euphoria, bipolar syndrome, anxiety, aphasia, dysphasia,fatigue, Uhthoff's symptom, gastroesophageal reflux, and sleepingdisorders.

Improvements in cognition outcomes associated with MS therapy, whetherslowing of cognitive decline, stabilization of cognitive decline, orimprovement of cognitive function, can be assessed using the PASAT(e.g., PASAT 2 or PASAT 3) or SDMT test, or alternatively the MS-COGtest (see Erlanger et al., J Neuro Sci 340: 123-129 (2014)). Thus, incertain embodiments, the methods of treatment disclosed herein includemethods for stabilizing or improving cognition in a patient, whereby thepatient's cognition outcome after one year of therapy is at least about10%, at least about 25%, at least about 40%, at least about 50%, or evenat least about 60% higher relative to a control patient not receivingthe estrogen/progestogen therapy (but otherwise receiving the sametreatment as the estrogen-treated patient), e.g., as measured by any ofthe preceding tests.

Improvements in memory associated with MS therapy, whether slowing ofmemory loss, stabilization of memory loss, or improvement of memory, canbe assessed using, for example, the 7/24 Spatial Recall Test. Thus, incertain embodiments, the methods of treatment disclosed herein includemethods for stabilizing memory loss or improving memory in a patient,whereby the patient's memory after one year of therapy is at least about10%, at least about 25%, at least about 30%, at least about 40%, atleast about 45%, at least about 50%, or even at least about 60% higherrelative to a control patient not receiving the estrogen/progestogentherapy (but otherwise receiving the same treatment as theestrogen-treated patient), e.g., as measured by the 7/24 Spatial RecallTest.

In some embodiments, substantial loss of memory refers to worseningperformance on the 7/24 Spatial Recall Test or Delayed Recall Test. Forexample, a subject who scores less than 7 on the Spatial Recall Test orDelayed Recall Test has substantial memory loss. Additionally, a subjectwho scores a 7 on the 7/24 Spatial Recall Test or Delayed Recall Test ona first date followed by a score of 6 or less on a subsequent date hassubstantial loss of memory over the period of time defined by the firstdate and the subsequent date.

While the various methods disclosed herein are typically efficaciouswhen administered without additional therapeutics, in certainembodiments, any of these methods further includes the step ofadministering to the subject an immunotherapeutic agent, wherein theimmunotherapeutic agent is neither an estrogen nor a progestogen. Thatis, in certain embodiments the subject is administered, in addition tothe estrogen, a second agent useful in the treatment of MS. In certainembodiments, the subject is administered, in addition to the estrogenand progestogen (or placebo), a third agent useful in the treatment ofMS. Such agents useful in the treatment of MS are, in general,immunotherapeutic agents. At least in connection with MS, such agentsare sometimes referred to as disease-modifying therapies ordisease-modifying therapeutics (DMTs).

In certain embodiments, the subject is already receiving adisease-modifying therapeutic. In this circumstance, the subject cancontinue to receive the disease-modifying therapeutic while taking theestrogen, with and without the progestogen. Significantly, however, thedose of the disease-modifying therapeutic may be decreased when used incombination with the estrogen, with and without the progestogen. Forexample, a current standard dose for glatiramer acetate (Copaxone®) is40 mg subcutaneously (s.c.) three times a week, or 20 mg s.c. daily. Inconjunction with estrogen and progestogen in accordance with theinvention, the dose for glatiramer acetate (Copaxone®) may be reduced byup to 50 percent or more, e.g., to 20 mg s.c. three times a week.

As another example, a current standard dose for fingolimod (Gilenya®) is0.5 mg by mouth (p.o.) daily. In conjunction with estrogen andprogestogen in accordance with the invention, the dose for fingolimod(Gilenya®) may be reduced by up to 50 percent or more, e.g., to 0.25 mgp.o. daily.

As another example, a current standard dose for dimethyl fumarate(Tecfidera®) is 240 mg p.o. daily. In conjunction with estrogen andprogestogen in accordance with the invention, the dose for dimethylfumarate (Tecfidera®) may be reduced by up to 50 percent or more, e.g.,to 120 mg p.o. daily.

As yet another example, a current standard dose for interferon beta-1a(Avonex® or Rebif®) is 30 μg intramuscularly (i.m.) weekly (Avonex®) or44 μg s.c. three days a week (Rebif®). In conjunction with estrogen andprogestogen in accordance with the invention, the dose for Avonex® maybe reduced to 15 μg i.m. weekly, and the dose for Rebif® may be reducedto 22 μg s.c. three days a week.

As yet another example, a current standard dose for interferon beta-1b(Betaseron® or Extavia®) is 0.25 mg s.c. every other day (Betaseron® orExtavia®). In conjunction with estrogen and progestogen in accordancewith the invention, the dose for interferon beta-1b (Betaseron® orExtavia®) may be reduced to 0.125 mg s.c. every other day.

In certain embodiments, the subject is receiving an immunotherapeuticagent and experiencing a relapse or progression of the multiplesclerosis. For example, a subject may experience a relapse orprogression while on a maintenance dose of a DMT. Such subject can thenbegin concurrent treatment with estrogen in accordance with any of thevarious methods disclosed herein, e.g., to reduce the frequency and/orseverity of relapses or to slow progression of the disease (e.g., asdetermined by assessment of one or more of walking, vision, balance,cognition, memory, or other symptoms of the condition, e.g., as measuredaccording to the Expanded Disability Severity Scale (EDSS) and/or themultiple sclerosis functional composite (MSFC)). Thus, the variousembodiments of the methods disclosed herein can be methods for improvingwalking, vision, balance, cognition, memory, or other symptoms in asubject, such as a subject with multiple sclerosis, and/or methods forimproving EDSS or MSFC scores in a subject, such as a subject withmultiple sclerosis.

In certain embodiments, the subject is receiving an immunotherapeuticagent and experiencing a relapse of the multiple sclerosis. For example,a subject may experience a relapse while on a maintenance dose of a DMT.Such subject can then begin concurrent treatment with estrogen inaccordance with a method of the present invention, e.g., to reduce thefrequency and/or severity of relapses.

In certain embodiments, the subject is receiving an immunotherapeuticagent selected from interferon-beta 1a, interferon-beta 1 b, glatirameracetate, natalizumab, mitoxantrone, fingolimod, teriflunomide, anddimethyl fumarate during a ramp-up period for dose of theimmunotherapeutic agent, e.g., the patient begins receiving theimmunotherapeutic and the estrogen therapy at the same time or at aboutthe same time (such as for patients who have not previously receivedtreatments for their disease). Advantageously, estrogen induces a rapidonset of therapeutic effect on MS, while commonly an immunotherapeuticagent such as interferon-beta 1a, interferon-beta-1b, glatirameracetate, natalizumab, mitoxantrone, fingolimod, teriflunomide, ordimethyl fumarate may take weeks to months to induce observableimprovements on some or all symptoms.

In certain embodiments, the subject is receiving glatiramer acetateduring a ramp-up period for dose of the glatiramer acetate. In othercertain embodiments, the subject is not already receiving adisease-modifying therapeutic.

In certain embodiments, the estrogen and the progestogen are formulatedseparately from one another, e.g., the subject receives the estrogen asa single formulation and the progestogen as a separate formulation. Fororal administration, a given dose of each formulation can comprise oneor more pills, tablets, capsules, or the like (i.e., unit doses). Forexample, an 8 mg dose of estriol can be administered as four 2 mgcapsules, and a 0.7 mg dose of norethindrone can be administered as asingle capsule, though preferably each dose is administered in a singleunit dose (e.g., one unit dose each for the estrogen and theprogestogen).

In certain embodiments, e.g., where a placebo is administered with theestrogen on days when progestogen is not administered, the estrogen andthe placebo are formulated separately from one another. For example, thesubject is administered the estrogen as a single formulation and theplacebo as a separate formulation. For oral administration, a given doseof each formulation can comprise one or more pills, tablets, capsules,or the like (i.e., unit doses). For example, an 8 mg dose of estriol canbe administered as four 2 mg capsules, and a placebo can be administeredas a single capsule.

When a given dose of any agent involves administration of more than asingle unit dose, e.g., four 2 mg capsules of estriol, the individualunit doses can be administered at essentially the same time, or they canbe administered at different times on a given day, provided the entiredaily dose is administered within a single day. For example, four 2 mgcapsules of estriol can be taken together essentially once a day, orthey may be taken two at a time twice a day, or they may be taken one ata time four times a day. Additional schedules are contemplated by theinvention, again provided the entire daily dose is administered within asingle day. While it may be preferable that the subject follow the sameschedule from one day to the next, such is not required, once againprovided the entire daily dose is administered within a single day.

When the estrogen and the progestogen are formulated separately, theycan be administered essentially simultaneously, or they can beadministered sequentially with respect to each other. For example, inone embodiment the subject is administered four 2 mg capsules of estrioland one 0.7 mg capsule of norethindrone essentially simultaneously. Inanother embodiment, the subject is administered estriol in divideddoses, e.g., two 2 mg capsules twice daily, and the progestogen isadministered essentially simultaneously with one of the divided doses ofestriol. In yet another embodiment, the subject is administered estriolin divided doses, e.g., two 2 mg capsules twice daily, and theprogestogen is administered at a separate time from either one of thedivided doses of estriol.

Similarly, when the estrogen and the placebo are formulated separately,they can be administered essentially simultaneously, or they can beadministered sequentially with respect to each other. For example, inone embodiment the subject is administered four 2 mg capsules of estrioland one placebo essentially simultaneously. In another embodiment, thesubject is administered estriol in divided doses, e.g., two 2 mgcapsules twice daily, and the placebo is administered essentiallysimultaneously with one of the divided doses of estriol. In yet anotherembodiment, the subject is administered estriol in divided doses, e.g.,two 2 mg capsules twice daily, and the placebo is administered at aseparate time from either one of the divided doses of estriol.

In certain embodiments, the estrogen and the progestogen are formulatedtogether. For oral administration, a given dose of each component,formulated together, can comprise one or more pills, tablets, capsules,or the like (i.e., unit doses). For example, an 8 mg dose of estriol anda 0.7 mg dose of norethindrone can be coformulated and administered asfour capsules, each containing 2 mg estriol and 0.0875 mg norethindrone,though preferably, where applicable, they are coformulated as one unitdose comprising both the estrogen and the progestogen.

In certain embodiments, e.g., where a placebo is administered with theestrogen on days when progestogen is not administered, the estrogen andthe placebo are formulated together. For oral administration, a givendose of each component, formulated together, can comprise one or morepills, tablets, capsules, or the like (i.e., unit doses). For example,an 8 mg dose of estriol and a placebo can be coformulated andadministered as four capsules, each containing 2 mg estriol and asuitable amount of placebo.

When a given dose of any coformulation of estriol and progestogen (orplacebo) involves administration of more than a single unit dose, e.g.,four capsules, each containing 2 mg estriol and 0.0875 mg norethindrone,the individual unit doses can be administered at essentially the sametime, or they can be administered at different times on a given day,provided the entire daily dose is administered within a single day. Forexample, four capsules, each containing estriol and progestogen (orplacebo) can be taken together essentially once a day, or they may betaken two at a time twice a day, or they may be taken one at a time fourtimes a day. Additional schedules are contemplated by the invention,again provided the entire daily dose is administered within a singleday. While it may be preferable that the subject follow the sameschedule from one day to the next, such is not required, once againprovided the entire daily dose is administered within a single day.

Clinically and in various embodiments, MS can be assessed and monitoredusing any of a number of structural (anatomical) and functional tests,including, without limitation: magnetic resonance imaging (MRI); PacedSerial Addition Test (PASAT); symbol digit modalities test (SDMT);expanded disability status score (EDSS); multiple sclerosis functionalcomposite (MSFC); 25-foot walk test; 9-hole peg test; low contrastvisual acuity; MS Quality of Life; Modified Fatigue Impact Scale; BeckDepression Inventory; 7/24 Spatial Recall Test; Benton Forms F & G;Buschke Selective Reminding Test; Verbal Paired Associates; Word ListGeneration. Recently, the PASAT test of cognitive function has comeunder criticism by some for its test-retest reliability and practiceeffect whereby one naturally improves over time with repeated testtaking. Polman C H et al., Neurology 74 Suppl 3: S8-15 (2010). In someembodiments, assessment of MacDonald dissemination in space and timefinds use in the present methods. For example, for dissemination inspace, lesion imaging, such as, by way of illustration, Barkhof-TintoreMR imaging criteria, may be used. For instance, the following criteriacan be evaluated: (1) at least one gadolinium-enhancing lesion or 9 T2hyperintense lesions; (2) at least one infratentorial lesion; (3) atleast one juxtacortical lesion; (4) at least 3 periventricular lesions;and (5) a spinal cord lesion. Such imaging criteria can optionally beused in combination with evaluation for immunoglobulin abnormalities inthe cerebrospinal fluid (CSF), for example. For dissemination in time,MR imaging can also be used. For example, if an MR imaging scan of thebrain performed at ≥3 months after an initial clinical eventdemonstrates a new gadolinium-enhancing lesion, this may indicate a newCNS inflammatory event, because the duration of gadolinium enhancementin MS is usually less than 6 weeks. If there are no gadolinium-enhancinglesions but a new T2 lesion (presuming an MR imaging at the time of theinitial event), a repeat MR imaging scan after another 3 months may beneeded with demonstration of a new T2 lesion or gadolinium-enhancinglesion. In various embodiments, any one or more of these structural(anatomical) and functional tests may be used in conjunction with thepresent invention (e.g., to assess the effectiveness of a disclosedtreatment method).

In some embodiments, patients having MS may be identified by criteria bythe workshop on the diagnosis of MS (Poser et al., Ann. Neurol. 13:227,1983). Briefly, under these criteria, an individual with clinicallydefinite MS has experienced two attacks and clinical evidence of eithertwo lesions or clinical evidence of one lesion and paraclinical evidenceof another, separate lesion. Definite MS may also be diagnosed byevidence of two attacks and oligoclonal bands of IgG in cerebrospinalfluid or by combination of an attack, clinical evidence of two lesionsand oligoclonal band of IgG in cerebrospinal fluid. The McDonaldcriteria can also be used to diagnose MS. (McDonald et al., 2001,Recommended diagnostic criteria for Multiple sclerosis: guidelines fromthe International Panel on the Diagnosis of Multiple Sclerosis, AnnNeurol 50:121-127). The McDonald criteria include the use of MRIevidence of CNS impairment over time to be used in diagnosis of MS, inthe absence of multiple clinical attacks.

In some embodiments, treatment can be deemed to be effective if there isa statistically significant difference in the rate or proportion ofexacerbation-free or relapse-free patients between a treated group and aplacebo (or control) group for either of these measurements. Inaddition, time to first exacerbation and exacerbation duration andseverity may also be measured. A measure of effectiveness as therapy inthis regard is a statistically significant difference in the time tofirst exacerbation or duration and severity in the treated groupcompared to control group. An exacerbation-free or relapse-free periodof greater than one year, 18 months, or 20 months is particularlynoteworthy. Clinical measurements include the relapse rate in one andtwo-year intervals, and a change in EDSS, including time to progressionfrom baseline of 1.0 unit on the EDSS that persists for six months. On aKaplan-Meier curve, a delay in sustained progression of disability showsefficacy. Other criteria include a change in area and volume of T2images on MRI, and the number and volume of lesions determined bygadolinium enhanced images as described herein and known in the art.

In various embodiments, the gray matter loss or gray matter atrophy orgray matter volume, or any assessment or evaluation steps may bemeasured using an imaging technique and/or surrogate marker. Assessmentof gray matter volume, and atrophy, can also be accomplished using MRI.Preferably, the MRI is able to distinguish between gray matter and whitematter of the brain. For example, SIENAX software can be used to segmentthe MR images into gray matter/white matter/CSF compartments.Alternatively or in addition, FMRIB's Integrated Registration andSegmentation Tool (FIRST) can be used to segment the caudate, putamen,and thalamus, and BrainSuite13 can be used to segment the cerebralcortex. In certain embodiments, the MRI is able to distinguish betweengray matter and white matter of the spinal cord. In various embodiments,the imaging technique is one or more of magnetic resonance imaging(MRI), 3T, axial T2, fast fluid-attenuated inversion recovery (FLAIR),double inversion recovery (DIR), phase-sensitive inversion recovery(PSIR), ultra high-field MRI, magnetization transfer imaging (MTI),T1-relaxometry, diffusion tensor imaging (DTI, including for examplemean diffusivity (MD), fractional anisotropy (FA), radial diffusivity(Dr) and axial diffusivity (Da)), proton magnetic resonance spectroscopy(MRS)) and related techniques. In various embodiments, any of thetechniques described in Brain (2002) 125 (8): 1676-1695, the contents ofwhich are hereby incorporated by reference in their entirety, can beused alone or in combination to, for example, measure brain volume, graymatter atrophy. For example, the following may be used: linear regionalmeasures; threshold-based measures; segmentation-based methods (e.g.,CSF volume, BPF, WBR, BICCR, Fuzzy connectedness segmentation, SPM-basedsegmentation); template driven segmentation of the brain; SIENAX);registration-based methods (e.g., MIDAS (Medical Image Display andAnalysis Software) method, voxel-based morphometry and brain surfacemodelling). Further, the present methods may employ any of the imagingmethods described in Am J Neuroradiol. (2011) 32(2): 408-12, thecontents of which are hereby incorporated by reference in theirentirety.

In various embodiments, the surrogate marker is one or more of thosedescribed in PLoS One 8(9): e6617 (September 2013) or U.S. Pat. No.8,772,225, the contents of which are hereby incorporated by reference intheir entirety. For example, in some embodiments, the surrogate markermay be one or more of nogo receptor, kallikrein-6 (neurosin),cerebellin-1, ceruloplasmin, dickkopf-3 (rig-like 7-1), amyloid betaprecursor-like protein 1, activated leukocyte cell adhesion molecule (CD166), neural cell adhesion molecule 2, neural epidermal growth factorlike 2/cerebral protein-12, clusterin (apolipoprotein j, complementlysis inhibitor), brevican, neuronal cadherin, chitinase-3-like 1protein, neogenin, multifunctional protein MFP (collagen alpha 1 (XVIII)chain, endostatin), dystroglycan 1, contactin 2, ephrin type a receptor4, neural cell adhesion molecule L1 like protein, and contactin 1.

In various embodiments, such measurements may be taken on a biologicalsample (e.g., cerebrospinal fluid (CSF), blood, a biopsy (e.g., frozentissue specimen, cultured cells, formalin-fixed paraffin-embedded tissuespecimen, etc.)). In some embodiments, the surrogate biomarker may beevaluated using one or more of mass spectrometry, immunohistochemicalstaining, western blotting, in cell western, immunofluorescent staining,ELISA, a radioimmunological assay (RIA), immunoblotting, a LINE blot,and fluorescent activating cell sorting (FACS). In some embodiments, thesurrogate biomarker may also be evaluated on a nucleic acid level (e.g.,low-to-mid-plex techniques, including but not limited to reporter geneassays, Northern blot, fluorescent in situ hybridization (FISH), andreverse transcription PCR (RT-PCR)) and higher-plex techniques,including but not limited to, serial analysis of gene expression (SAGE),DNA microarrays, Tiling array, RNA-Seq/whole transcriptome shotgunsequencing (WTSS), high-throughput sequencing, multiplex PCR, multiplexligation-dependent probe amplification (MLPA), DNA sequencing byligation, and Luminex/XMAP).

In some aspects, the present invention relates to a method of treatingneurodegenerative disease (e.g., one or more forms of MS), in a patienthaving a decrease of white matter fA (fractional anisotropy) of about10% (or about 5%, or about 6%, or about 7%, or about 8%, or about 9%, orabout 10%, or about 11%, or about 12%, or about 13%, or about 14%, orabout 15%), comprising administering to the patient an effective amountof an estrogen, which is optionally estriol and, optionally any one ofthe other therapies disclosed herein (e.g., one or more progestogens(e.g., progesterone)) at any of the doses and regimens described herein.In some aspects, the present invention relates to a method of preventingor reversing white matter fA loss in a patient in need thereof,comprising administering to the patient an effective amount of anestrogen, and, optionally any one of the other therapies disclosedherein (e.g., one or more progestogens (such as progesterone)) at any ofthe doses and regimens described herein. In some embodiments, whitematter fA is measured using any of the methods described herein (e.g.,imaging techniques and/or surrogate biomarkers), including, by way ofexample, diffusion tensor imaging (DTI). In various embodiments, whitematter may be measured in various substructures including, for example,the optic nerve and cervical spinal cord. In various embodiments, thedecrease of white matter fA, or prevention or reversal of white matterfA loss, is in one or more white matter substructures including, forexample, the optic nerve and cervical spinal cord.

As described in the Examples below, a randomized, double-blind,placebo-controlled clinical trial was designed to ascertain whethertreatment with an estrogen pill (estriol), used in combination with anFDA-approved standard treatment for MS (Copaxone®) for two years, canreduce brain gray matter atrophy as compared to treatment with a placebopill in combination with the same major FDA-approved standard treatmentfor MS. Results from this study are presented in Example 1 below.Briefly, results from this study showed a reduction in total GM atrophyby adding estriol treatment as compared to placebo treatment to standardtherapy.

Data in Example 1 are from the completed trial, as above, withCopaxone®) (glatiramer acetate) injections (20 mg/day) started at month0.

Also as described in the Examples below, an ongoing randomized,double-blind, placebo-controlled clinical trial was designed toascertain whether, in women, treatment with an estrogen pill (estriol),used in combination with major FDA-approved standard treatments for MS(Betaseron®, Extavia®, Rebif®, Avonex®, Copaxone®, Gilenya®, Aubagio®,or Tecfidera®) for one year, can improve cognitive testing as comparedto treatment with a placebo pill in combination with the same majorFDA-approved standard treatments for MS. Interim results from thisongoing study are presented in Example 2 below.

With respect to the estriol intervention, the study design includescontinuous treatment with estriol, part of the time with norethindrone,and part of the time without norethindrone. That is, again with respectto the estriol intervention, the study can be understood as a series ofconsecutive periods, wherein for each period the subject continuouslyreceives estriol and, for only a portion of each period, the subjectalso receives norethindrone.

In the experimental group, subjects receive standard MS treatment plusestriol 8 mg by mouth daily (continuously) plus norethindrone 0.7 mg bymouth daily for two weeks starting at month 6 and at months 9 and 12.

In the control group, subjects receive standard MS treatment plusestriol placebo by mouth daily (continuously) plus norethindrone placeboby mouth daily for two weeks starting at month 6 and at months 9 and 12.

Study subjects are 18- to 50-year-old women with diagnosis of clinicallydefinite or MacDonald criteria relapsing-remitting MS,secondary-progressive MS, or primary-progressive MS; on a stable dose ofCopaxone®, Betaseron®, Extavia®, Rebif®, Avonex®, Gilenya®, Aubagio®, orTecfidera® for a minimum of 3 months duration prior to enrollment; andwith no relapse within 30 days before trial enrollment. Excluded fromthe study are women on oral contraceptives (OCP), hormone replacementtherapy (HRT), progesterone intrauterine devices (IUDs), or other sexhormones.

In the ongoing study, the primary outcome measure is change frombaseline in cognitive function (processing speed), assessed by PacedSerial Addition Test (PASAT). Numerical test scores (ranging from 0-60)are acquired, then percent change for each subject at trial conclusionas compared to baseline is determined. A primary goal is to determinewhether greater improvement as expressed as percent change occurs in theestriol group as compared to the placebo group.

Secondary outcome measures in the ongoing study include change frombaseline in cognitive function as assessed by cognitive evokedpotentials, measured in milliseconds; change from baseline in standardMS outcome measures (relapses, expanded disability status score (EDSS),25-foot walk test, 9-hole peg test, low contrast visual acuity, MSQuality of Life, Modified Fatigue Impact Scale, and Beck DepressionInventory); change from baseline in cognitive function as assessed by abrief battery of cognitive tests; and safety.

Cognitive evoked potentials are recorded in msecs for each subject atbaseline and conclusion. The percent improvement at conclusion ascompared to baseline for each subject is determined. Group comparisonswill reveal whether the percent improvement is greater in the estrioltreated group as compared to the placebo treated group.

A brief battery of cognitive tests is administered, including:Processing speed: symbol digit modalities test (SDMT); Visual memory:7/24 Spatial Recall Test, Benton Forms F & G; Verbal memory: BuschkeSelective Reminding Test, Verbal Paired Associates; and Language: WordList Generation. Each subject is tested at baseline, month 6, andconclusion. Percent change at conclusion as compared to baseline isdetermined in each subject. Group comparisons will reveal whichcognitive test within the battery had greater improvement in the estrioltreated group as compared to the placebo treated group.

Safety is measured based on neurologic exams, laboratory tests(chemistries, complete blood count (CBC)), and breast and gynecologicexams. Having now described the present invention in detail, the samewill be more clearly understood by reference to the following examples,which are included herewith for purposes of illustration only and arenot intended to limit the invention.

EXAMPLES Example 1—Use of Copaxone® and Estriol for the Treatment ofMultiple Sclerosis

Sixteen sites randomized subjects 1:1 to oral estriol (8 mg daily) ororal placebo for 24 months (FIG. 1). A four week taper commenced atmonth 24 for both estriol and placebo. To avoid taking unopposedestrogens, the Estriol+GA subjects also received a progestin (0.7 mgnorethindrone) daily for two weeks duration every three months startingat month 6, and Placebo+GA received a second placebo for progestin. Allstarted GA injections (20 mg/day per day) within 2 months ofrandomization. Randomization had one stratification factor: GA treatmentduring screening. Each study site had separate examining and treatingneurologists unaware of assignment. The examining neurologists performedneurologic assessments including EDSS, while treating neurologistsmanaged patient care including treatment of relapses. 158 subjects wereenrolled.

Each subject had a brain MRI done at month 0, 3, 6, 12, 24. The effectof estriol treatment as compared to placebo treatment on total (whole)brain gray matter (GM) atrophy was assessed since gray matter atrophy isa surrogate marker for permanent disability in MS. SIENAX software wasused to segment the MR images into gray matter/white matter/CSFcompartments from 10 estriol+Copaxone® (E+C) and 10 placebo+Copaxone®(P+C) subjects who had completed all 24 months of treatment. The GMvolume change from the month 0 scan to the month 12 scan and then thevolume change from the month 0 scan to the month 24 scan was calculated.

The GM volume change after 12 months was −9.5±3.8 mL (or −1.22±0.49%) inplacebo+Copaxone® patients, and −1.0±2.0 mL (or −0.13±0.26%) inestriol+Copaxone® patients (p=0.071).

The GM volume change after 24 months was −12.5±5.0 mL (or −1.61±0.65%)in placebo+Copaxone® patients, and −2.0±4.4 mL (or −0.27±0.58%) inestriol+Copaxone® patients (p=0.138).

Thus the overall rate of GM atrophy in the placebo plus Copaxone®treated group was over 1.0% per year, while in the estriol plusCopaxone® treated group over the same period there was no significantatrophy. This represented a reduction in GM atrophy by adding on estrioltreatment as compared to placebo treatment to standard therapy.

The effects of estriol treatment on various brain GM regions were thenassessed. FMRIB's Integrated Registration and Segmentation Tool (FIRST)was applied to segment the caudate, putamen, and thalamus, andBrainSuite13 to segment the cerebral cortex, from five subjects in theE+C group and five subjects from the P+C group. The volume change ineach structure from the month 0 scan to the month 12 scan and then thevolume change from the month 0 scan to the month 24 scan was calculatedand plotted (FIG. 2). E+C patients demonstrated a trend towards lessatrophy than P+C patients at both time points in all four structures.This data on less GM substructure volume loss with estriol treatment isconsistent with the observation (above) of less whole GM volume losswith estriol treatment.

Example 2—Use of Copaxone® and Estriol for the Treatment of MultipleSclerosis

This example describes a randomized, double-blind, placebo-controlledhuman clinical trial for the treatment of multiple sclerosis usingCopaxone® and estriol.

Enrollment Criteria

Eligible patients were females, an age of 18-50 years, a diagnosis ofrelapsing remitting multiple sclerosis as defined according to theMcDonald criteria (Polman C. et al., Neurology 64:987 (200)), a baselinescore of 0 to 4.5 on the Expanded Disability Status Scale (EDSS, whichranges from 0 to 10, with higher scores indicating more severedisability), and disease activity as evidenced by at least twodocumented relapses in the previous 24 months before screening or asevidenced by at least one documented relapse within 24 months beforescreening with a history of at least one gadolinium-enhancing lesion ona brain or cord magnetic resonance imaging (MRI) scan performed at least3 months before or 3 months after the clinical relapse. Key exclusioncriteria were progressive forms of multiple sclerosis, other clinicallysignificant diseases, pre-specified laboratory test abnormalities,possible malignancy on mammogram or uterine ultrasound, exposure toglatiramer acetate for longer than 2 months before randomization,relapse or steroid use within 30 days prior to randomization, use of anyinterferon, ACTH, corticosteroids, intravenous immunoglobulins, or otherlisted MS treatments within 2 months before screening, those who werepregnant, breastfeeding, or trying to get pregnant, those not willing todiscontinue other hormonal treatments, those who underwent surgical ornatural menopause for longer than 1 or 3 years, respectively, with nohormone replacement therapy, those who had ever been treated with amajor immunosuppressive contraindicated treatment.

TABLE 1 Baseline Characteristics of the Intention-to-Treat Population.*Estriol + GA Placebo + GA Patient Characteristics (N = 82) (N = 76)Age - yr 37.7 ± 7.6  37.1 ± 7.3  Race - no. (%)† Caucasian 65 (79.3) 62(81.6) Black 9 (11.0) 7 (9.2) Hispanic 7 (8.5) 6 (7.9) Other 1 (1.2) 1(1.2) Time since diagnosis - yr 3.3 ± 4.6 2.9 ± 4.5 Number of previousrelapses Within 1 yr before screening 1.5 ± 0.7 1.5 ± 0.7 Within 2 yrbefore screening 2.0 ± 0.7 2.3 ± 0.9 Prior GA treatment Never 25 (30.5)27 (35.5) Previously 17 (20.7) 6 (7.9) During screening 40 (48.8) 43(56.6) Prior treatment with any interferon - no. (%)‡ No 59 (72.0) 50(65.8) Yes 23 (28.0) 26 (34.2) Mean score on EDSS¶ 2.2 ± 1.2 2.1 ± 1.1EDSS sore at baseline - no. (%)¶ 0 9 (11.0) 6 (7.9) 1.0 or 1.5 16 (19.5)21 (27.6) 2.0 or 2.5 27 (32.9) 24 (31.6) 3.0 or 3.5 25 (30.5) 22 (29.0)4.0 4 (4.9) 2 (2.6) 5.5 1 (1.2) 1 (1.3) Gadolinium-enhancing lesionsnumber 1.0 ± 2.3 0.9 ± 2.0 Active lesions on brain MRI - no. (%) No 55(67.9) 53 (70.7) Yes 26 (32.1) 22 (29.3) Volume of lesions on T2weighted 6.8 ± 8.9  7.7 ± 11.2 Images - cm³ *Plus-minus values are means+/− SD. All patients were included as the intention-to-treat populationwho underwent randomization, except those with no data afterrandomization. There were no significant differences between baselineclinical or demographic characteristics between the study groups. †Racewas self-reported. ‡Patients may have received more than one priormultiple sclerosis medication. Patients may have received othernon-approved therapies for multiple sclerosis before enrollment in thestudy. The percentage of patients receiving medication for multiplesclerosis before study entry was balanced across treatment groups.¶Scores on the Expanded Disability Status Scale (EDSS) ranged from 0 to10, with higher scores indicating a greater degree of disability. Thebaseline EDSS score was higher than inclusion criteria of 4.5 in twopatients (EDSS = 5.5), one in each study group that were 4.5 at firstscreening visit, but 5.5 at baseline. One patient in the Estriol + GAgroup did not have a confirmed relapse within 24 months prior torandomization, with enrollment based on disease activity evidenced byMRI enhancing lesions.Efficacy Measures

Standardized neurologic assessments, including an EDSS assessment, wereperformed at months 0, 3, 6, 12, 18 and 24, and at the time of asuspected relapse (as an additional unscheduled visit). EDSS assessmentswere performed by physicians who were trained either by in-persontraining or online (www.Neurostatus.net). MRI scans were obtained atscreening and at months 0, 3, 6, 12 and 24. Subjects were seen orcontacted every 3 months for compliance assessments and for dispensingmedications.

The primary efficacy end point was the annualized relapse rate. Arelapse was defined as the appearance of new neurological symptoms orthe worsening of pre-existing symptoms, lasting at least 48 hours in asubject who had been neurologically stable or improving in the previous30 days, accompanied by an objective change in a neurologicalexamination (i.e., a worsening of 0.5 or more points on the EDSS or aworsening by 1.0 or more points on the pyramidal, cerebellar, brainstemor visual functional system scores, not due to fatigue alone and notassociated with fever or infection). The treating physician made thedecision concerning whether the relapse criteria had been met,incorporating whether a change in EDSS had been documented by theexamining physician. Both treating and examining physicians were unawareof study group assignments. The standard treatment for relapse was a 3-5day course of glucocorticoids at the discretion of the treatingneurologist.

Secondary efficacy end points included the proportion of subjects with arelapse over all 24 months, a change in PASAT cognitive testing, asustained improvement in PASAT cognitive testing (as defined by anincrease of at least 3 points sustained over at least 6 months), achange in EDSS scores from baseline, disability progression (as definedby an increase in EDSS of at least 1.0 point in subjects with a baselinescore of 1.0 or higher, or by an increase of at least a 1.5 points insubjects with a baseline score of 0, each sustained for at least 6months). Tertiary end points included gray matter atrophy on MRI.

Study Conduct and Monitoring Schedule

Patients were randomized to Copaxone® (glatiramer acetate) injections(20 mg/day) and oral estriol (8 mg/day) or to Copaxone® injections andplacebo for a 24-month treatment duration. Gynecologists examined thepatients before, during, and after the study. Each patient was examinedat three- to six-month intervals during the trial. Patients alsounderwent mammograms before and after the study. In addition, atbaseline, three months, six months, 12 months, 18 months, and 24 months,the investigators measured participants' estriol levels, and assessedfor MS relapses and MS-related disabilities.

A total of 82 patients received Copaxone® plus estriol, and 76 patientsreceived Copaxone® plus placebo. Baseline characteristics were similarin both patient groups. Participants' mean age at entry wasapproximately 38, and their mean EDSS score at entry was 2.2. Estriollevels in serum were in a mid-pregnancy range in the estriol-treatedgroup. To ensure breast and uterus safety, every three months thepatients took norethindrone 0.7 mg once a day for 14 days. This hormoneregimen was found to be safe and well tolerated with regard to seriousadverse events, adverse events, general exams, blood chemistries, andhematological studies, as well as for gynecological outcomes (see Table2). Irregular menses occurred more with Estriol+GA (P<0.001), whilevaginal infections occurred more with Placebo+GA (P<0.05), with noincrease in discontinuations due to either.

TABLE 2 Adverse Events and Serious Adverse Events Estriol + GA Placebo +GA Adverse Events† (N = 82) (N = 76) Any adverse event - no. of events,[no of pts, % of 480 [76, 93%] 392 [67, 87%] pts] Most frequent events -no. of events [no of pts, % of pts] Copaxone injection areaabnormalities 51 [26, 32%]* 30 [14, 18%] Upper respiratory infection 33[22, 27%] 38 [26, 34%] Irregular menses/spotting 26 [19, 23%]*** 4 [3,4%] Urinary tract infection 23 [15, 18%] 16 [10, 13%] Fatigue 15 [13,16%] 10 [8, 10%] Depression/anxiety 14 [12, 15%] 10 [9, 12%] Menstrualflow amount increased 12 [11, 13%] 8 [6, 8%] Headache 11 [9, 11%] 12[11, 14%] Nausea/vomiting 9 [7, 9%] 5 [5, 6%] Sinusitis 6 [6, 7%] 14[10, 13%]* Arm/leg numbness, tingling 7 [6, 7%] 10 [7, 9%]Gastroenteritis 7 [5, 6%] 4 [3, 4%] Dizziness 5 [4, 5%] 10 [7, 9%]Vision problem (blurry, double) 6 [4, 5%] 7 [7, 9%] Back pain 5 [4, 5%]5 [5, 6%] Menstrual cramp 4 [4, 5%] 5 [4, 5%] Insomnia 4 [4, 5%] 4 [4,5%] Heart palpitation 2 [2, 2%] 4 [4, 5%] Shingles 7 [2, 2%] 4 [4, 5%]Vaginal infection 1 [1, 1%] 9 [8, 10%]** Adverse events leading todiscontinuation - no. (%) 5 (6%) 5 (6%) Severe adverse events‡ 9 [8,10%] 12 [10, 13%] MS relapse 2 [2, 2%]¶ 6 [5, 6%]¶ Pregnancy termination2 [2, 2%] 0 UTI 1 [1, 1%] 1 [1, 1%] Migraine headache related eye pain 1[1, 1%] 0 Heart failure 1 [1, 1%] 0 Pace maker implantation 1 [1, 1%] 0Pyelonephritis 1 [1, 1%] 0 Systolic heart failure 1 [1, 1%] 0 Accidentlytook other's drug 0 1 [1, 1%] Acute appendicitis 0 1 [1, 1%] B-celllymphoma§ 0 1 [1, 1%] Car accident related body numbness 0 1 [1, 1%]Right knee replacement 0 1 [1, 1%] Other safety events monitored UterusEndometrial thickness >8 mm (ultrasound) - no. 24 (29) 27 (36) (%)Endometrial biopsies performed§§ - no. (%) 9 (11) 6 (8) Fibroids(ultrasound) - no. (%) 8 (10) 8 (11) Abnormal proliferation on biopsy -no. (%) 0 0 Breast Fibrocystic disease on clinical exam 5 (6) 4 (5)Mammogram with malignancy 0 0 †All patients who took at least one doseof study drug were included. However, among the 6 patients who droppedshortly after baseline visit, five did not have safety evaluation dataand were excluded from the safety analysis. The listed events reportedby % were rounded up to nearest integer. The events are listed bydecreasing incidence in the Estriol + GA group, within each category.*AE significantly higher in one treatment group compared to the other;***indicating P <0.001, **indicating P < 0.05, and *indicating P < 0.10.‡SAE patients were all hospitalized, but none had severe or immediatelylife-threatening condition. §This patient, in the placebo group,discontinued the study at the time of B-cell lymphoma diagnosis when wason study for 12 months and died 17 months later. ¶In Estriol + GA group,both patients discontinued the study: 1 before and 1 after Month 12. InPlacebo + GA group, 3 patients discontinued the study: 1 before and 2after Month 12. §§Four patients had multiple uterine endometrialbiopsies: two patients had two biopsies each in the Estriol + GA groupand two patients had three biopsies each in the Placebo + GA group. Noabnormal proliferation was found. Note: No laboratory abnormalitiesoccurred significantly more frequently in either treatment group.

Safety assessments, including clinical, blood laboratory safety testingand assessments of estriol levels, occurred at months 0, 3, 6, 12, 18,and 24. On study blood tests included complete blood count (CBC) withdifferential and platelets; chemistry panel including sodium, potassium,creatinine, BUN, glucose, total protein, albumin, bilirubin (total),alkaline phosphatase, AST (SGOT), and ALT (SGPT), and lipid profile(HDL, LDL and triglycerides, cholesterol. Gynecologic exams were done atmonth 0, 6, 18 and at month 24 exit, with uterine ultrasounds at months6, 18 and at month 24 exit. Mammograms were done in screening and atmonth 24 exit. Adverse event analysis was based on the percentage ofpatients who discontinued the study and the percentage of patients whodiscontinued the study possibly due to adverse events.

Statistical Analysis

The sample size was determined based on the primary end point ofannualized relapse rate. A total sample of 150 eligible patients wouldprovide approximately 80% power at a two-sided significance level of0.10 for this phase II clinical trial to detect the difference in theannualized relapse rate of 0.76 vs 1.18 for Estriol plus GA group andthe Placebo plus GA group in 2 years.

Intention-to-treat analyses were carried out for all end points. For theprimary endpoint, a negative binomial regression model was used tocompare both 12 months and 24 months annualized relapse rates betweenEstriol+GA versus Placebo+GA groups adjusted for covariates. To controlthe overall type I error, a sequential testing procedure was applied. Ahierarchical statistical approach was used whereby results in the first12 months of treatment would be assessed, and, if and only if,significance were met, results in the entire 24 months of treatmentwould be assessed. The earlier timepoint was compared first since GArequires time to reach full efficacy, potentially providing a greaterwindow to detect efficacy 12 months after initiation of GA and studydrug treatment. Consistent with a phase 2 study using a clinicaloutcome, a p-value<0.10 was considered statistically significant.

For the time to first relapse analysis, Kaplan Meier curves and log-ranktest were used to estimate and compare the relapse free probabilities ofthe two treatment groups. Cox proportional hazards model was used tocompare the time to relapse free probabilities between two groupsadjusting for covariates. The fixed effects include treatment groups(Estriol+GA vs Placebo+GA), baseline lesion number, age and baselineEDSS score. The random effect of subject is included in the model toaccount for within subject correlation.

Linear mixed effects model was carried out to compare the percent changein whole gray matter and cortical gray matter between treatment groups.For the exploratory endpoints of EDSS, PASAT, and brain volume measures,linear mixed effects model was used to compare treatment groups at 12and 24 months. Linear mixed effects model was carried out to evaluatethe association between PASAT change and percent brain volume change.

Multiple imputation on the missing data was also performed according tothe pattern mixture model as a sensitivity analysis. The pattern mixturemodel provides the analysis with the possibility of non-random dropout.The missing data were sequentially imputed by the follow up time and theimputation model assumed that the treatment effect for patients afterdrop out is the same as taking placebo.

Patients

A total of 164 patients were randomized, of which 158 received studydrug and had at least one visit thereafter (intention-to-treatpopulation). Of the 158 patients, 82 were assigned to the Estriol+GAgroup and 76 to the Placebo+GA group (FIG. 1). Baseline demographics anddisease characteristics were well balance across both patient groups(Table 1).

The rate of discontinuation was similar between groups (FIG. 1). A totalof 60 patients (73.2%) in the Estriol plus GA group and 56 (73.7%) inthe Placebo plus GA group completed the 24 month study treatmentduration. Of the 158 patients, 15.8% discontinued the study during thefirst year (7.6% in the Estriol plus GA group and 8.2% in the Placeboplus GA group), and an additional 10.7% discontinued the study duringthe second year (6.3% and 4.4%, respectively). Reasons fordiscontinuation did not differ between groups. The most common reasonsfor discontinuation were lost to follow up or patient's decision basedon family issues or time constraints.

Primary Outcome Measure

The primary outcome measure for disease efficacy was annualized relapserate. While most Phase II trials used surrogates or biomarkers as theprimary outcome, the trial focused on an outcome measure acceptable forapproval by the FDA. Since this was a Phase II trial, it was powered toreduce relapses by one third more in the Copaxone® plus estriol group ascompared to the Copaxone® plus placebo group, with a targeted p value ofp=0.10 at the end of study which was 24 months. As shown in FIG. 3,after 24 months of treatment the primary outcome measure was attained byreducing relapse rates by 32% (p=0.11) in the Copaxone® plus estriolgroup as compared to the Copaxone® plus placebo group. Surprisingly,after only 12 months of treatment, the relapse rate was reduced by 47%(p=0.02) in the Copaxone® plus estriol group as compared to theCopaxone® plus placebo group, see FIG. 3. In the entire 24 months oftreatment, the relapse rate was reduced by 32% (P=0.098) in the Estriolplus GA group as compared to the Placebo plus GA group. Thus, inaddition to finding that Copaxone® plus estriol treatment hadsignificant benefit in reducing the frequency of relapses over 24months, the combination treatment also had a more rapid onset of actionas compared to Copaxone® plus placebo.

Regarding temporal patterns, relapse rates remained low and unchangedfrom month 12 (0.25) to month 24 (0.25) with Estriol+GA, while relapserates decreased gradually from month 12 (0.48) to month 24 (0.37) withPlacebo+GA. A more rapid onset of efficacy with Estriol+GA was alsoobserved when examining the proportion of subjects relapse free over 24months, with differences beginning at 6-12 months, favoring Estriol+GA,P=0.096.

These results were surprising given that estriol treatment was notcompared to a true placebo, but rather was tested in combination withstandard-of-care therapy (Copaxone®).

Since anti-inflammatory drugs the FDA has approved have so far requiredmuch larger sample sizes to show a significant reduction in relapserates, even as compared to a true placebo, the results of the studyadding estriol to Copaxone® suggest a novel mechanism of action, amechanism never before observed in MS. We hypothesize, based on oursurprising findings, that this novel mechanism of action may entaildirect effects of estriol treatment on preserving gray matter (total aswell as substructure) volumes.

TABLE 3 Clinical End Points* Estriol + GA Placebo + GA End Point (n =82) (n = 76) Annualized relapse rate in 12 months Rate (95% CI)† 0.25(0.16-0.40) 0.48 (0.33-0.69) Adjusted rate ratio E + GA vs. P + GA (95%CI)§ 0.51 (0.29-0.90)*¹ Annualized relapse rate in 24 months Rate (95%CI)† 0.25 (0.17-0.37) 0.37 (0.25-0.53) Adjusted rate ratio E + GA vs.P + GA (95% CI)§ 0.65 (0.39-1.08)*² Time to first confirmed relapseProportion of pts with relapse at 12 months 22.8 (15.0-33.7) 33.1(23.5-45.2) % (95% CI)‡ Proportion of pts with relapse at 24 months 33.3(23.8-45.4) 42.9 (32.1-55.5) % (95% CI)‡ Adjusted hazard ratio E + GAvs. P + GA (95% 0.63 (0.36-1.09)*³ CI)¶ Time to disability progressionProportion of pts with progression at 24 11.4 (5.9-21.7) 15.8 (8.8-27.6)months % (95% CI)‡ Adjusted hazard ratio E + GA vs. P + GA (95% 0.81(0.32-2.07) CI)¶ EDSS score reduction from baseline to Month 24 Mean ±SD, Median 0.29 ± 0.98, 0.5 0.05 ± 1.13, 0.0 *Plus-minus values aremeans ± SD. CI denotes confidence interval, E + GA for Estriol + GA, andP + GA for Placebo + GA. †Annualized relapse rates were calculated basedon negative binomial regression. §Relapse rate ratio was estimated usingnegative binomial regression with adjustment for age, baseline EDSS (<2vs. ≥2), number of relapse 12 months prior study entry (0-1 vs. >1), MSduration (<1 vs. ≥1 year), prior GA treatment (never vs. past/current),and prior interferon treatment (yes vs. no). ‡Values were calculatedusing the Kaplan-Meier product-limit method. Progression defined as EDSSincrease of at least 1.0 point in subjects with baseline score of 1.0 orhigher or increase of at least 1.5 points with baseline score of 0, eachsustained for at least 6 months. ¶Hazard ratio was estimated using Coxproportional hazard regression. For relapse, age, baseline EDSS (<2 vs≥2), number of relapse 12 months prior study entry (0-1 vs. >1), MSduration (<1 vs. ≥1 year), prior GA treatment (never vs, past/current),and prior interferon treatment (yes vs. no) were adjusted; for EDSSprogression, age and baseline EDSS (<2 vs. ≥2) were adjusted. *¹P =0.021; *²P = 0.098; *³P = 0.096Disability Assessment

Exploratory disability outcomes revealed promising trends forimprovement in the Estriol plus GA group. The Expanded Disability StatusScale (EDSS) is a standard composite disability score used extensivelyin MS trials. Higher scores indicate worse disability. This compositecovers a variety of disabilities (including ambulation, vision,cognition, coordination, etc.), but the scoring is not linear and thecomposite score is understood to be principally an indicator of thelevel of disability in ambulation. While there was no change in the EDSSscores for the Copaxone® plus placebo treatment group, the Copaxone®plus estriol treatment group showed a significant decrease (i.e.,improvement) in this disability score (FIG. 6). The probability ofdisability worsening or EDSS progression (as defined by an increase inEDSS of 1 point for over 6 months) was 15.8% for the Placebo plus GAgroup, and 11.4% for the Estriol plus GA group (Table 3). EDSS scoreswere then assessed for possible improvement with combination treatment.While EDSS scores in the Placebo plus GA group were stable and unchangedover the entire 24 month treatment duration, the Estriol plus GA groupshowed a significant improvement in EDSS scores by the end of study,month 24, with a median change in EDSS of a half step (EDSS absolutemedian change=−0.5, p=0.03), however group differences in EDSSimprovement were not powered for significance (FIG. 7A).

Another clinical disability measure with treatment effects was the timed25-foot walk test. This test measures how many seconds it takes to walk25 feet, with higher scores indicating worse disability. The walk timewas significantly increased in the Copaxone® plus placebo group(p=0.03), while it was slightly decreased in the Copaxone® plus estriolgroup, together resulting in a significant between-group difference(p=0.02). Together these data show a gradual worsening in walking timesin the Copaxone® plus placebo treated group, which did not occur in theCopaxone® plus estriol treated group. This beneficial effect of estrioltreatment on 25-foot walking times is consistent with the beneficialeffect of estriol treatment on EDSS scores since the latter is weightedtoward being an indicator of ambulation.

There were no significant differences between groups in the MultipleSclerosis Functional Composite (MSFC), which reflects a composite ofscores including the Paced Auditory Serial Addition Test (PASAT) forcognition, the 9 hole peg test and the 25 foot walk test (Table 4).However, an interesting effect of combination treatment was observed oncognitive disability. A perfect PASAT score is 60, with scores lowerthan 55 serving as a continuous variable for disability. By 12 months oftreatment, PASAT scores improved significantly as compared with scoresat baseline, by approximately 6% (i.e., 3 points) among patientsreceiving Estriol plus GA, while no significant improvement was observedin those receiving Placebo plus GA, (p=0.04 between group difference,all adjusted for covariates of age, education and baseline scores).Subgroup analysis showed that this improvement in PASAT scores in theEstriol plus GA group at month 12 was due to improvements in those withmore cognitive disability at baseline (FIG. 4). After 12 months oftreatment, patients receiving Copaxone® plus estriol continued to havehigh PASAT scores to the end of study at month 24, while participantsreceiving placebo began to show improved PASAT scores by month 24.Notably, a change of six or more points in tests of processing speed inMS is considered to be clinically significant. Further, Copaxone® plusestriol treatment improved function in those with significant cognitivedisability, rather than merely slowing cognitive decline. Thisrepresents repair of disability, not merely prevention of worsening.This beneficial effect on PASAT scores at 12 months in the Estriol plusGA group could not be attributed to practice effects of repeated testingsince the comparison was with the Placebo plus GA group tested atidentical time points.

Next it was shown that the improvement in PASAT cognitive test scoreswas sustained when subjects were followed for the entire 24 monthperiod, p=0.02 (FIG. 5).

In addition, the beneficial effects of estriol treatment on cognitivefunction were shown using another cognitive test, the 7/24 spatialrecall test (for spatial memory). While initial encoding of informationdid not differ between groups, the number of subjects with perfectscores for immediate recall (p=0.006) and delayed recall (p=0.04) washigher in the Copaxone® plus estriol treated group as compared to theCopaxone® plus placebo treated group over the entire 24 month treatmentduration. Such rapid and potent effects on cognition that were observedin the Copaxone® plus estriol group as compared to the Copaxone® plusplacebo group were surprising and point to a novel effect on cognitivedisability not seen before with other FDA-approved MS drugs.

TABLE 4 Multiple Sclerosis Functional Composite (MSFC) Estriol + GAPlacebo + GA End Point (N = 82) (N = 76) MSFC Score^(a) Baseline score(Mean ± SD, Median) −0.04 ± 0.69, 0.05  0.06 ± 0.79, 0.24 Change frombaseline at Month 12 N = 70 N = 58 Mean ± SD, Median 0.13 ± 0.37    0.06± 0.38    Change from baseline at Month 24 N = 60 N = 54 Mean ± SD,Median 0.10 ± 0.35    0.09 ± 0.43    PASAT3 score - All Patients^(a)Baseline score (Mean ± SD, Median) 51.0 ± 8.9, 55   52.3 ± 9.1, 56  change from baseline at Month 12 N = 70 N = 61 Mean ± SD, Median  1.9 ±5.6, 1.0** 0.1 ± 4.5, 0   change from baseline at Month 24 N = 60 N = 55Mean ± SD, Median 1.1 ± 4.0, 1.0 1.1 ± 4.3, 0   % change from baselineat Month 12 N = 70 N = 61 Mean ± SD, Median (%)   5.5 ± 15.6, 1.8** 0.8± 9.9, 0   % change from baseline at Month 24 N = 60 N = 55 Mean ± SD,Median (%)  2.9 ± 10.6, 1.7 2.7 ± 10.3, 0  PASAT3 score - Patients withbaseline score <55^(a) N = 39 N = 33 Baseline score (Mean ± SD, Median)43.7 ± 7.6, 45   44.7 ± 9.1, 49   change from baseline at Month 12 N =33 N = 25 Mean ± SD, Median  4.7 ± 6.6, 4.0* 1.6 ± 6.0, 1.0 change frombaseline at Month 24 N = 26 N = 23 Mean ± SD, Median 2.3 ± 5.2, 3.5 3.0± 5.9, 4.0 % change from baseline at Month 12 N = 33 N = 25 Mean ± SD,Median (%)  12.6 ± 19.9, 7.6*  4.0 ± 14.0, 3.8 % change from baseline atMonth 24 N = 26 N = 23 Mean ± SD, Median (%)  6.4 ± 14.8, 6.8  6.9 ±14.7, 9.1 9-Hole Peg Test^(b) Baseline value (Mean ± SD, Median) 19.7 ±3.8, 19.0 19.1 ± 2.7, 18.8 Change from baseline at Month 12 N = 70 N =63 Mean ± SD, Median −0.6 ± 1.5, −0.6 −0.1 ± 1.8, −0.4 Change frombaseline at Month 24 N = 60 N = 56 Mean ± SD, Median −0.1 ± 3.5, −0.6−0.4 ± 1.5, −0.5 25-foot Walk Time^(b) Baseline value (Mean ± SD,Median) 4.9 ± 1.0, 4.7 4.9 ± 1.5, 4.5 Change from baseline at Month 24 N= 70 N = 60 Mean ± SD, Median 0.1 ± 0.8, 0.1 0.0 ± 0.9, 0.0 Change frombaseline at Month 24 N = 60 N = 55 Mean ± SD, Median −0.1 ± 0.6, −0.00.1 ± 1.1, 0.1 ^(a)Change from baseline positive value indicateimprovement for MSFC and PASAT3 ^(b)Change from baseline positive valueindicate worsening for 9-Hole Peg test and 25-Foot Walk Time. **P <0.05, student t-test comparing the means of the two study groups *P <0.10, student t-test comparing the means of the two study groups §Values were calculated using the Kaplan-Meier product-limit method.

In contrast to month 12 observations, absolute PASAT scores were nodifferent at month 24 in the Estriol plus GA group compared to thePlacebo plus GA group (FIG. 7B; Table 4). This was due to both a trendfor improvement in the Placebo plus GA group as well as a trend forworsening in the Estriol plus GA group. To address whether a trend forworsening in the Estriol plus GA group at month 24 might be related tothe decrease in estriol levels at month 24, correlations between estriollevels and improvement in PASAT scores were assessed. Indeed, higherestriol levels correlated with greater improvement in PASAT scores(p=0.03 for all patients; p=0.07 for Estriol+GA patients only).

Gray matter volumes, specifically cortical gray matter volumes, havepreviously been associated with cognitive test scores. There was lesscortical gray matter atrophy (45%) and whole gray matter atrophy (30%)at month 12 in the Estriol plus GA group compared to the Placebo plus GAgroup (cortical gray matter: Estriol+GA=−0.41, Placebo+GA=−0.74,p=0.079; whole gray matter: Estriol+GA=−0.47, Placebo+GA=−0.68, p=0.139)(FIGS. 7C & 7D). This gray matter sparing was independently confirmedusing voxel-based morphometry (VBM), the latter revealing which graymatter regions were preserved with Estriol+GA compared to Placebo+GA(FIGS. 7 and 9). Similar to effects on PASAT scores, beneficial effectson gray matter sparing in the Estriol+GA group were no longer present atmonth 24. Indeed, correlations between PASAT improvement and gray mattersparing were found (cortical gray matter, p=0.0327; whole gray matter,p=0.0359), which was present in the Estriol+GA group (cortical graymatter, p=0.0159; whole gray matter, p=0.0093) and absent in the controlPlacebo+GA group. In contrast, the Estriol+GA group compared to thePlacebo+GA group had more white matter atrophy.

TABLE 5 MRI Volumes Estriol + GA Placebo + GA Comparing the two (N = 82)(N = 76) study groups§ Enhancing Lesion Volume^(a) Baseline 79.7 ± 220,0   54.2 ± 126, 0   (Mean ± SD, Median) Change from baseline at Mon 12 N= 69 N = 62 Mean ± SD, Median −51.2 ± 202, 0   −18.7 ± 184,0    −12.5(−69.0-44.1)  p = 0.665 Change from baseline at Mon 24 N = 55 N = 55Mean ± SD, Median −39.3 ± 196, 0   −34.0 ± 120, 0   −1.6 (−62.6-59.5) p= 0.960 Number of Enhancing Lesions^(a) Baseline (Mean ± SD, Median) 1.0± 2.3, 0   0.9 ± 2.1, 0   Change from baseline to Mon 12 N = 68 N = 62Mean ± SD, Median −0.9 ± 2.2, 0    −0.5 ± 1.8, 0    0.89, (0.54-1.45)  p = 0.631 Change from baseline to Mon 24 N = 55 N = 55 Mean ± SD, Median−0.9 ± 2.5, 0    −0.5 ± 2.4, 0    0.89, (0.54-1.48)   p = 0.655 Lesionactivity on Brain MRI - proportion of patients with enhancing lesionBaseline %  32.1 (21.9-42.3) 29.3 (19.0-39.6) (95% CI) Month 12 % 14.5(6.2-22.8) 21.0 (10.8-31.1) 0.30 (0.07-1.31)  (95% CI) p = 0.110 Month24 % 14.6 (5.2-23.9) 14.6 (5.2-23.9)  0.66 (0.13-3.40)  (95% CI) p =0.616 Brain Volume - All Patients Baseline (Mean ± SD, Median) Wholebrain 1604 ± 62, 1607   1602 ± 51, 1602   Whole gray matter 954 ± 51,957   926 ± 52, 967   Cortical gray matter 754 ± 46, 755   761 ± 42,762   White matter 650 ± 35, 650   640 ± 37, 635   % change frombaseline (Mean ± SD, Median)^(b) Whole Brain at Month 12 −0.50 ± 0.70,−0.49 −0.50 ± 0.64, −0.43 −0.00 (−0.23-0.23)  p = 0.988 Month 24 −0.89 ±0.82, −1.0  −0.78 ± 0.73, −0.91 −0.02 (−0.26-0.22)  p = 0.877 Whole graymatter at Month 12 −0.48 ± 0.82, −0.50 −0.69 ± 0.71, −0.68 0.21(−0.05-0.47) p = 0.108 Month 24 −0.96 ± 0.75 −0.94, −0.95 ± 0.76, −0.930.12 (−0.16-0.39) p = 0.411 Cortical gray matter at Month 12 −0.44 ±0.92, −0.51 −0.72 ± 0.80, −0.67 0.29 (−0.01-0.58) p = 0.056 Month 24−0.96 ± 0.86, −0.97 −1.04 ± 0.87, −0.93 0.23 (−0.09-0.54) p = 0.156White matter at Month 12 −0.51 ± 1.06, −0.16 −0.20 ± 0.83, −0.11 −0.29(−0.63-0.04)  p = 0.090 Month 24 −0.77 ± 1.28, −0.66 −0.54 ± 1.12, −0.56−0.20 (−0.56-0.15)  p = 0.261 Plus-minus values are means ± SD. CIdenotes confidence interval, E + GA indicates Estriol + GA, and P + GAindicates Placebo + GA. ^(a)Change from baseline negative valueindicates improvement. ^(b)Change from baseline negative value indicatesworsening. §For Volume of Enhancing Lesions and Brain Volume (wholebrain, gray matter, cortical gray matter and white matter,respectively), linear mixed effect model was developed using allfollow-up data to estimate the difference of the value change frombaseline between the two study groups at Months 24 and 12 while baselinevalue was adjusted.

-   -   For Number of Enhancing Lesions, the data in this column are        E+GA vs. P+GA mean of lesions number ratio (95% CI) and p-value        based on mixed effect negative binomial regression model.    -   For Lesion Activity on Brain MRI, the data in this column are        E+GA vs. P+GA odds ratio (95% CI) and p-value based on mixed        effect logistic regression model.        MRI Methodologies

MRI scans were performed at 0, 3, 6, 12 and 24 months using astandardized protocol implemented at each site that consisted of thefollowing: T1-weighted 3D volume, pre and post contrast: TR2200, TE3.4,TI 900, 176 slices, 1 mm³. Dual-echo fast spin echo: TR10000, TE12/95,50 slices, 1×1×3 mm. Fluid attenuated inversion recovery (FLAIR):TR10380, TE88, T188, T12500, 50 slices, 1×1×3 mm. Minor changes wereallowed to accommodate different platforms and field strengths at eachsite. MRI data in Dicom format were fully anonymized prior to transferand then uploaded to the central MRI reading center database. Prior tostudy onset, each site provided a dummy scan utilizing the standardizedsequences for review by the central MRI reading center to verify scanquality and fidelity. Quality control was maintained at each site usingstandard procedures for clinical scanners (daily phantoms, stabilitytesting). Quarterly phantoms were collected from 12 of the 15 sites,most using the standard American College of Radiology (ACR) phatom. Onesite upgraded from a Siemens 1.5T to a 3.0T in November 2013, resultingin the acquisition of one month 24 scan on the new scanner. One siteupgraded from a Phillips Achieva 3.0T to a Pillips Intera 3.0T after thefirst subject completed month 24. All subsequent studies were performedon the Intera.

Scans underwent a standard review locally by a radiologist blind tostudy details to assess for any new or unusual findings as a safetymeasure. Incoming imaging data was reviewed for completeness andfidelity to study pulse sequences by the imaging core investigators.Local radiologists and imaging core investigators were all blind torandomization assignment. All MRI investigators remained blinded totreatment assignment until the end of the study.

MRI brain, whole gray matter, whole white matter and cortical graymatter volumes were determined using a pairwise Jacobian integration(PJI) method. Pre-processing for structural T1-weighted imagesincluded 1) N3 non-uniformity correction, 2) histogram-based intensitynormalization, 3) linear standard space registration using ICBM 2009cnonlinear symmetric template, 4) patch-based brain extraction, and 5)lesion-inpainting. Inputs to PJI were a pair of baseline and follow-uppre-processed structural T1-weighed images. The PJI consisted of 1)linear skull-constrained symmetric registration, 2) halfwaytransformation and resampling, 3) nonlinear symmetric registration usingANTS, and 4) voxelwise Jacobian determinant calculation on the warpfield. Whole gray matter and whole white matter tissue masks weregenerated by SPM8 Segment function. Additional nonlocal means denoisingwas applied. For whole brain tissue masks, the whole gray matter andwhole white matter masks were combined. For cortical gray matter mask, astandard cortical mask was nonlinearly transformed and merged with graymatter mask. The standard template was the ICBM (ICBM 2009c nonlinearsymmetric version), and the nonlinear registration was performed byANTS. Finally, the Jacobian determinants were averaged within the masksfor percent volume change in cortical gray matter, whole gray matter,whole white matter, and whole brain.

Voxel-based morphometry (VBM) analyses were performed as described byKurth et al. (Neuroimage Clin. 4:454 (2014)). All subjects included inthe VBM cohort were required to have at least reached month 12 of thestudy, and all images had to pass quality control before and after imagepreprocessing to be included in the VBM cohort. Using this criteria, theVBM cohort consisted of Ill subjects (62 in the estriol+GA, and 49 inthe placebo+GA group) from 13 sites for month 12 analyses, and 86 ofthese subjects (45 in the estriol+GA, and 41 in the placebo+GA group)for 24 month analyses.

Brain images were preprocessed utilizing SPM8 and the VBM8 toolbox.White matter lesions were in-painted to minimize their impact based onmanual delineations that were used for the analysis of new T2 lesions.For this purpose, these manually delineated lesion masks werecoregistered to the T1-weighted images, corrected if necessary, and usedfor lesion in-painting as described by Chard et al. (J. Magn. Reson.Imaging 34:223 (2010)). The lesion in-painted images were subsequentlyrealigned for each subject using halfway-registrations and corrected forbias-field inhomogeneities. The realigned, bias corrected images werethen tissue-classified into gray matter, white matter, and cerebrospinalfluid and registered to MNI space through linear and non-linearlytransformations (see http://dbm.neuro.uni-jena.de/vbm8/VBM8-Manual.pdf).More specifically, the tissue classification was based on maximum aposteriori segmentations, accounted for partial volume effects, and wasrefined by applying a spatially adaptive non-local means denoisingfilter as well as a hidden Markov random field model. These methods madethe tissue-classification independent of tissue probability maps andthus additionally minimized the influence of misclassifications,lesions, and altered geometry. Using DARTEL, the gray matter segmentswere then spatially normalized to the DARTEL Template supplied with theVBM8 Toolbox (see http://dbm.neuro.uni-jena.de/vbm), resulting in avoxel-wise comparability between subjects and time-points. Finally, thegray matter segments were smoothed with a Gaussian kernel (8 mm fullwidth at half maximum). These smoothed gray matter segments constitutedthe input for the statistical analysis. For visualization, a meantemplate was created from the normalized brain images of all subjects,allowing the results from the statistical analysis to be related to theunderlying mean anatomy of the subject sample.

VBM Statistical Analyses.

For the statistical analysis, a general linear model was applied thatused the smoothed gray matter segments as the dependent and group x timeas the independent variable. Subject and scan site were added asvariables of no interest, thus effectively controlling forinter-individual differences (e.g. individual anatomy, age, diseaseduration, etc.) as well as the potentially confounding impact ofdifferent scanners. Non-sphericity was modeled and accounted for asdescribed previously and implemented in SPM8. Applying this model, theinteraction between group and time was calculated using T-tests toinvestigate group differences in local gray matter changes between month0 and month 12 (month 0 and month 24, respectively). In addition, thegray matter loss within each group was investigated by calculatingT-tests for month 0>month 12 (month 0>month 24, respectively) for eachgroup separately. All results were corrected for multiple comparisons bycontrolling the false discovery rate (FDR) using a threshold of P≤0.05.Corrected results were rendered on the mean template of all subjects inFIG. 7. In addition, significant findings were visualized using maximumintensity projections as shown in FIG. 9.

Cognitive Testing

The estriol and placebo subjects were administered the 7/24 SpatialRecall Test periodically throughout the study. A perfect score on thetest is 7, and for both treatment groups, the median score was also 7.Therefore, performance was assessed by the proportion of subjects ineach group who achieved a perfect score on the test. The estriol groupperformed statistically better than the placebo group (Table 6).Logistic regression for repeated measurement was used to compare theprobability having a perfect score between the two treatment groups,adjusting for age, education level, and baseline recall (Table 7).

TABLE 6 Percentage of subjects achieving a perfect score on the 7/24Spatial Recall Test Month Estriol + GA Placebo + GA 0 65.8 (54/82) 71.1(54/76) 3 72.5 (58/80) 60.8 (45/74) 6 70.5 (55/78) 73.2 (52/71) 12 74.3(52/70) 71.4 (45/63) 18 88.9 (56/63) 63.6 (35/55) 24 76.3 (45/59) 75.0(42/56)

TABLE 7 Odds ratio for achieving a perfect score on the 7/24 SpatialRecall Test Estimated perfect Estriol vs. Placebo score rate (95% CI)Odds ratio Estriol (%) Placebo (%) Odds ratio (95% CI) P-value 80.6(74.9, 85.2) 69.8 (63.4, 75.5) 1.79 (1.18, 2.73) P = 0.0067

The estriol and placebo subjects were administered the Delayed RecallTest periodically throughout the study. A perfect score on the test is7, and for both treatment groups, the median score was also 7.Therefore, performance was assessed by the proportion of subjects ineach group who achieved a perfect score on the test. The estriol groupperformed statistically better than the placebo group (Table 8).Logistic regression for repeated measurement was used to compare theprobability having a perfect score between the two treatment groups,adjusting for age, education level, and baseline recall (Table 9).

TABLE 8 Percentage of subjects achieving a perfect score on the DelayedRecall Test Month Estriol + GA Placebo + GA 0 67.1 (55/82) 71.1 (54/76)3 75.0 (60/80) 65.9 (48/74) 6 73.1 (57/78) 76.1 (54/71) 12 68.6 (48/70)69.8 (44/63) 18 85.7 (54/63) 65.4 (36/55) 24 79.7 (47/59) 75.0 (42/56)

TABLE 9 Odds ratio for achieving a perfect score on the Delayed RecallTest Estimated perfect Estriol vs. Placebo score rate (95% CI) Oddsratio Estriol (%) Placebo (%) Odds ratio (95% CI) P-value 81.8 (75.5,86.8) 73.2 (65.7, 79.5) 1.65 (1.01,2.70) P = 0.0455

Example 3—Use of Copaxone® and Estriol for the Treatment of MultipleSclerosis

Alternatively or in addition to all of the foregoing as it relates togray matter, the invention further contemplates that white matter fA(fractional anisotropy) can be employed in a manner analogous to thegray matter atrophy as discussed herein in various embodiments.

Diffusion Tensor Imaging (DTI) assesses white matter, specifically whitematter tract integrity. A decrease in fA can occur with eitherdemyelination or with axonal damage or both. One can assess white mattersubstructures including optic nerve and cervical spinal cord.

MRIs of brain including high cervical spinal cord to be done at month 6,1 year, and 2 years. If a decrease in fA of 10% is observed in fA of 2tracts, treat with estriol to halt this decrease. Alternatively if fA isdecreased by 10% in only one tract but that tract is associated withclinical deterioration of the disability served by that tract, treatwith estriol. Poorer scores in low contrast visual acuity wouldcorrelate with decreased fA of optic nerve, while poorer motor functionwould correlate with decreased fA in motor tracts in cervical spinalcord.

INCORPORATION BY REFERENCE

All patents, published patent applications, and other publicationsmentioned in the description above are incorporated by reference hereinin their entirety.

EQUIVALENTS

Having now fully described the present invention in some detail by wayof illustration and example for purposes of clarity of understanding, itwill be obvious to one of ordinary skill in the art that the same can beperformed by modifying or changing the invention within a wide andequivalent range of conditions, formulations and other parameterswithout affecting the scope of the invention or any specific embodimentthereof, and that such modifications or changes are intended to beencompassed within the scope of the appended claims. As used herein,methods of treatment are equally applicable to uses of a compositiondescribed herein for treating the diseases or disorders described hereinand/or compositions for use and/or uses of a composition describedherein in the manufacture of a medicaments for treating the diseases ordisorders described herein.

I claim:
 1. A method of slowing or halting cortical gray matter atrophyin a human subject suffering from multiple sclerosis comprisingadministering orally to a subject in need thereof, on a continuous basisfor a treatment period of 84 consecutive days (12 weeks), 8 mg ofestriol daily; administering orally to the subject, for 14 consecutivedays (2 weeks) of the treatment period, 0.7 mg of norethindrone daily;and wherein prior to administering the estriol the subject is receivingan immunotherapeutic agent or an anti-inflammatory agent.
 2. The methodof claim 1, further comprising administering to the patient animmunotherapeutic agent.
 3. The method of claim 2, wherein theimmunotherapeutic agent is selected from interferon-beta 1a,interferon-beta 1b, glatiramer acetate, natalizumab, mitoxantrone,fingolimod, teriflunomide, dimethyl fumarate, mycophenolate mofetil,paclitaxel, cyclosporine, corticosteroids, azathioprine,cyclophosphamide, methotrexate, cladribine, 4-aminopyridine, andtizanidine.
 4. The method of claim 2, wherein the immunotherapeuticagent is glatiramer acetate.
 5. The method of claim 4, wherein the isglatiramer acetate is administered subcutaneously to the subject on acontinuous basis for the treatment period.
 6. The method of claim 5,wherein the glatiramer acetate is subcutaneously administered to thesubject in an amount of 20 mg daily.
 7. The method of claim 1, whereinprior to administering the estriol the subject is receiving animmunotherapeutic agent.
 8. The method of claim 1, wherein the methodfurther comprises the steps of: assessing the change in the subjectscortical gray matter volume over a period of time; and administering theestriol to the subject in addition to the immunotherapeutic agent or ananti-inflammatory agent if the cortical gray matter volume decreases byat least 0.3 percent between a first assessment and a second assessmentabout 6 months after the first assessment, by at least 0.6 percentbetween a first assessment and a second assessment about one year afterthe first assessment, or by at least 1.0 percent between a firstassessment and a second assessment about two years after the firstassessment.
 9. The method of claim 8, wherein the first assessment isperformed before, at the same time as, or at about the same time as thesubject receives the immunotherapeutic agent or anti-inflammatory agent.10. The method of claim 8, wherein the second assessment is performed atleast about six months, at least about one year, at least about 18months, or at least about two years after said first assessment.
 11. Themethod of claim 1, wherein the method comprises four consecutivetreatment periods, five consecutive treatment periods, six consecutivetreatment periods, seven consecutive treatment periods, or eightconsecutive treatment periods.
 12. The method of claim 11, wherein afterfour treatment periods the subject's gray matter atrophy is less than1.5%, less than 1.4%, less than 1.3%, less than 1.2%, less than 1.1%,less than 1.0%, less than 0.9%, less than 0.8%, less than 0.7%, lessthan 0.6%, or less than 0.5% as compared to the start of administration.13. The method of claim 11, wherein after four treatment periods thesubject's gray matter atrophy is reduced by 3.5% as compared to asubject who has not been administered the estriol and norethindrone. 14.The method of claim 1, wherein the multiple sclerosis isrelapsing-remitting multiple sclerosis.
 15. The method of claim 1,wherein the multiple sclerosis is secondary-progressive multiplesclerosis.
 16. The method of claim 1, wherein the multiple sclerosis isprimary-progressive multiple sclerosis.
 17. The method of claim 1,wherein the multiple sclerosis is progressive-relapsing multiplesclerosis.
 18. The method of claim 1, wherein the estriol andnorethindrone are formulated together.
 19. The method of claim 1,wherein the subject is experiencing progression of the multiplesclerosis.
 20. The method of claim 1, wherein the subject hasprogressive walking disability.